Substituted organosulfur compounds and methods of using thereof

ABSTRACT

The present invention provides substituted di-, tri-, tetra- and penta-sulfide compounds and compositions, and methods of using the same for the treatment and/or prevention of a cell proliferative disorder. The present invention also provides methods for preparing trisulfide compounds and compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a DIVISIONAL of U.S. patent application Ser. No.11/110,203, filed Apr. 20, 2005, which claims the benefit of priorityunder 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/564,151,filed Apr. 20, 2004: the contents of each of these applications areincorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to organosulfur compounds and methods of usingthereof.

BACKGROUND ART

Cancer remains one of the most important unmet medical challenges tomankind. A number of options for treating tumors are available,including surgery, radiation, chemotherapy, or any combination of theseapproaches. Among these, chemotherapy is widely used for all types ofcancers, in particular for those inoperable or with metastaticcharacteristics. Despite a variety of chemotherapeutic compounds beingused in clinics, chemotherapy is generally not curative, but only delaysdisease progression. Commonly, tumors and their metastasis becomerefractory to chemotherapy, as the tumor cells develop the ability ofmultidrug resistance. In some cases, the tumors are inherently resistantto some classes of chemotherapeutic agents. In other cases, the acquiredresistance against chemotherapeutic agents is developed during thechemotherapeutic intervention. Thus, there remain significantlimitations to the efficacy of available chemotherapeutic compounds intreating different classes of tumors. Furthermore, many cytotoxic andcytostatic agents used for chemotherapeutic treatment of tumors havesevere side effects, resulting in termination of the chemotherapy insome patients. Thus, there remains a need for new chemotherapeuticagents.

Dibenzyl trisulfide (DBTS) is a biologically active polysulfidesecondary metabolite that was isolated from the sub-tropical shrub,Petiveria alliacea L. (Phytolaccaceae). It has been reported that DBTShas immunomodulatory activities (“Immunomodulatory activities ofPetiveria alliacea.”, by Williams, L. A. D., Gardner, T. L., Fletcher,C. K., Naravane, A., Gibbs, N. and Fleischhacker, R. Phytother. Res.,1997, 11, 251-253; “A sulfonic anhydride derivative from dibenzyltrisulphide with agro-chemical activities”, by Williams, L. A. D.,Vasquez, E., Klaiber, I., Kraus, W. and Rosner, H. Chemosphere, 2003,51, 701-706). In investigating the cellular and molecular mechanisms ofDBTS for its immunomodulatory activity, Rosner and co-workers reportedthat DBTS preferentially binds to an aromatic region of bovine serumalbumin and attenuates the dephosphorylation of tyrosyl residues of MAPkinase (erk1/erk2) in SH-SY5Y neuroblastoma cells (in “Disassembly ofmicrotubules and inhibition of neurite outgrowth, neuroblastoma cellproliferation, and MAP kinase tyrosine dephosphorylation by dibenzyltrisulphide”, by Rosner, H., Williams, L. A. D., Jung, A. and Kraus, W.Biochim. Biophy. Acta, 2001, 1540, 166-177). In addition, they reportedthat DBTS causes a reversible disassembly of microtubules and did notaffect actin dynamics in SH-SY5Y neuroblastoma cells and in Wistar 38human lung fibroblasts. Furthermore, they reported that DBTS alsoinhibits neuroblastoma cell proliferation and neurite outgrowth fromspinal cord explants.

In a different study, Mata-Greenwood and co-workers tested theantiproliferative and differentiating activity of a large set ofextracts derived from various plants (“Discovery of novel inducers ofcellular differentiation using HL-60 promyeolocytic cells”, byMata-Greenwood, E., Ito A., Westernburg, H., Cui, B., Mehta, R. G.,Kinghorn, A. D. and Pezzuto, J. M. Anticancer Res. 2001, 21, 1763-1770).They reported that the lipophilic extract of the roots of Petiveriaalliacea L., and the active fraction from the lipophilic extract showedantiproliferative and differentiating activity in HL-60 promyelocyticcells. From the active fraction of the lipophilic extract, they isolatedtwo active organosulfur compounds, i.e., 2-[(phenylmethyl)dithio]ethanoland dibenzyl trisulfide. They reported that these two organosulfurcompounds induced monocyte-like differentiation and strong cytotoxicity.Furthermore, they reported that none of these two isolates demonstratedantiproliferative activity in HL-60 cells.

DISCLOSURE OF THE INVENTION

The present invention relates to organosulfur compounds, pharmaceuticalcompositions, and methods of using thereof. More particularly, thepresent invention relates to substituted di-, tri-, tetra- andpenta-sulfide compounds, including pharmaceutically acceptable salts andpartially oxidized sulfone derivatives thereof. Compounds as describedherein exhibit anti-tumor, anticancer, anti-inflammation,anti-infectious, and/or antiproliferation activity. The presentinvention also relates to methods of making and formulating organosulfurcompounds.

In one embodiment, the invention provides compounds having formula

wherein A and B are the same or different, and are independently anoptionally substituted aryl, heteroaryl, or a 5-14 membered ring whichmay be monocyclic or multicyclic and optionally containing a heteroatom;

each S is optionally in the form of an oxide;

S¹ and S² are independently S, SO or SO₂;

each R is H, halogen, carboxyl, cyano, amino, amido, an amino acid, aninorganic substituent, SR¹, OR¹ or R¹, wherein each R¹ is alkyl,alkenyl, alkynyl, aryl, heteroaryl, a carbocyclic ring or a heterocyclicring, each of which is optionally substituted and may contain aheteroatom;

m, n and p are independently 0-3;

or a compound having formula (3) or (4):

wherein A, B, R, S, n and p are as defined above;

or a compound having formula (5):

wherein A, B, S, n and p are as defined above; and

Z is (CR¹ ₂)_(q) or (CR¹═CR¹)_(q)* wherein q is 0-3 and the * representsthat C═C may be replaced with alkynyl, O, S, NR; or Z is an optionallysubstituted aryl, heteroaryl or heterocyclic ring;

wherein A and B together may form a cyclic ring system;

and a pharmaceutically acceptable salt, ester, prodrug or metabolitethereof;

provided said compound is not dibenzyltrisulfide,di(p-chlorobenzyl)trisulfide, (p-chlorobenzyl)benzyltrisulfide,di(p-nitrobenzyl)trisulfide, di(3-phenyl-2-propenyl)-trisulfide,diphenyltrisulfide, or di(p-t-butylphenyl)trisulfide.

In the above formula 1-5, each Z may be

wherein each W is independently a bond, CR, N, NR, S, or O;

each R is as defined above.

In the above formula 1-5, each R may be H, halo, OR¹, SR¹, CO₂R¹, CONR¹₂, C═O, CN, CF₃, OCF₃, NO₂, NR₁R₁, OCOR₁; or R is C₁₋₁₀ alkyl, C₃₋₁₀cyclic alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, an aryl, heteroaryl, acarbocyclic ring or a heterocyclic ring, each of which may contain aheteroatom.

In the above formula 1-5, each A and B may be benzene, pyridine,pyridazine, pyrimidine, pyrazine, triazine, isoxazole, isothiazole,oxadiazole, [1,2,4]oxadiazole, triazole, thiadiazole, pyrazole,imidazole, thiazole, oxazole, benzoxazole, pyrrole, furan, thiopheneindolizine, indole, isoindole, indoline, benzofuran, benzothiophene,indazole, benzimidazole, benzthiazole, purine, quinoxaline, quinoline,isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline,naphthyridine, pteridine, acridine, phenazine, phenothiazine, indene,naphthalene, benzoxadiazol, or benzo[1,2,5]-oxadiazole.

In another aspect, each A and B are independently

where X and W are independently S, O, NR₇, CR₇;

or one W in a 6-membered monocyclic or bicyclic ring may be a bond; and

each R₁, R₂, R₃, R₄, R₅, R₆, R₇ is H, halogen, carboxyl, cyano, amino,amino acid, amido, an inorganic substituent, SR¹, OR¹ or R¹, whereineach R¹ is alkyl, alkenyl, alkynyl, aryl, heteroaryl, a carbocyclic ringor a heterocyclic ring, each of which is optionally substituted and maycontain a heteroatom. For example, each R₁, R₂, R₃, R₄, R₅, R₆, R₇ maybe H, halo, OR¹, SR¹, CO₂R¹, CONR¹ ₂, C═O, CN, CF₃, OCF₃, NO₂, NR₁R₁,OCOR₁; or each R₁, R₂, R₃, R₄, R₅, R₆, R₇ is C₁₋₁₀ alkyl, C₃₋₁₀ cyclicalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, an aryl, heteroaryl, a carbocyclicring or a heterocyclic ring, each of which may contain a heteroatom.

Examples of aryl, heteroaryl, or heterocyclic ring include but are notlimited to piperazine, piperidine, morpholine, thiomorpholine, phenyl,furanyl, thiophenyl, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl,quinoxalinyl, thiazolyl, oxazolyl, imidazolyl, quinolinyl, naphthalenyl,pyridazinyl, pyrazolopyrimidinyl, benzoimidazolyl, benzothiazolyl,benzene-thiophene, pyrazolyl, pyrrolyl, indolyl, isoindolyl,quinolizinyl, quinolinyl, isoquinolinyl, or quinazolinyl, each of whichis optionally substituted with a heteroatom selected from O, N, S andhalo; or substituted with C₁₋₁₀ alkyl, C₃₋₁₀ cyclic alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, aryl, or heterocycle, each optionally containinga heteroatom.

In the above formula 1-5, each S may be a mono-oxide or a di-oxide.

In another aspect, the compound has the formula (6)

and each n is 1-3; and

R is H, halo, alkyl or halogenated alkyl.

In yet another aspect, the compound has the formula (7)

wherein Ar is an optionally substituted thiophene, benzothiophene,pyridine or pyrazine. Examples of compounds having formula 1-5 includebut are not limited to di(fluorobenzyl)trisulfide,di(o-chlorobenzyl)trisulfide, di(methylbenzyl)trisulfide,di(trifluoromethylbenzyl)trisulfide, di(2-phenylethyl)trisulfide,di(2-thiophen-yl-methyl)trisulfide, di(4-pyridin-yl-ethyl)trisulfide,di(2-pyrimidin-yl-ethyl)trisulfide, ordi(3-benzothiophen-yl-methyl)trisulfide. In particular examples, thecompound is di(p-fluorobenzyl)trisulfide, di(m-methylbenzyl)trisulfide,or di-(p-methylbenzyl)trisulfide.

In another embodiment, the present invention provides methods for makinga composition comprising a compound having formula 1-5 as describedabove, and also provides compositions prepared according to suchmethods. In one aspect, the present invention provides a methodcomprising: a) dissolving a compound of claim 1 in a water-solubleorganic solvent, a non-ionic solvent, a water-soluble lipid, acyclodextrin, a vitamin, a fatty acid, a fatty acid ester, aphospholipid, or a combination thereof, to provide a solution; and b)adding saline or a buffer containing 1-10% carbohydrate solution. Theorganic solvent may be polyethylene glycol (PEG), an alcohol,N-methyl-2-pyrrolidone, N,N-dimethylformamide, N,N-dimethylacetamide,dimethyl sulfoxide, or a combination thereof.

In the above process, the non-ionic surfactant may bepolyoxyethyleneglyceroltriricinoleat 35, PEG-succinate, polysorbate 20,polysorbate 80, polyethylene glycol 660 12-hydroxystearate, sorbitanmonooleate, poloxamer, ethoxylated persic oil, capryl-caproylmacrogol-8-glyceride, glycerol ester, PEG 6 caprylic glyceride,glycerin, glycol-polysorbate, or a combination thereof. Particularexamples of non-ionic surfactants are polyethylene glycol modifiedCREMOPHOR® (polyoxyethyleneglyceroltriricinoleat 35), CREMOPHOR® EL,hydrogenated CREMOPHOR® RH40, hydrogenated CREMOPHOR® RH60, SOLUTOL® HS(polyethylene glycol 660 12-hydroxystearate), LABRAFIL® (ethoxylatedpersic oil), LABRASOL® (capryl-caproyl macrogol-8-glyceride), GELUCIRE®(glycerol ester), and SOFTIGEN® (PEG 6 caprylic glyceride).

In the above process, the lipid may be a vegetable oil, a triglyceride,a plant oil, or a combination thereof. For example, the lipid may becastor oil, polyoxyl castor oil, corn oil, olive oil, cottonseed oil,peanut oil, peppermint oil, safflower oil, sesame oil, soybean oil,hydrogenated vegetable oil, hydrogenated soybean oil, a triglyceride ofcoconut oil, palm seed oil, and hydrogenated forms thereof, or acombination thereof.

In the above process, the vitamin may be tocopherol; and the fatty acidand fatty acid ester may be oleic acid, a monoglyceride, diglyceride, amono- or di-fatty acid ester of PEG, or a combination thereof.

In the above process, the cyclodextrin may be alpha-cyclodextrin,beta-cyclodextrin, hydroxypropyl-beta-cyclodextrin, or sulfobutylether-beta-cyclodextrin. The phospholipid may be soyphosphatidylcholine, or distearoyl phosphatidylglycerol, andhydrogenated forms thereof, or a combination thereof. Furthermore, thecarbohydrate in the above process may comprise dextrose.

In yet another embodiment, the present invention provides methods forpreparing a compound of formula 1-2 as described above, comprising: a)contacting N-trimethylsilyl imidazole with sulfur dichloride in ahalogenated solvent to provide diimidazolylsulfide; and b) contactingsaid diimidazolylsulfide with mercaptan. In one example, the halogenatedsolvent is dichloromethane.

In one aspect, N-trimethylsilyl imidazole in hexane is contacted withsulfur dichloride in dichloromethane. In another aspect, sulfurdichloride as a neat compound is contacted with N-trimethylsilylimidazole in hexane and dichloromethane. In yet another aspect, themethods further comprise recrystallizing the trisulfide. In one example,the trisulfide is recrystallized in n-hexanes, hexanes, heptane,petroleum ether or a combination thereof.

In another embodiment, the present invention provides a pharmaceuticalcomposition comprising a compound having formula 1-5 as described above,and a pharmaceutically acceptable excipient. Such compounds andpharmaceutical compositions thereof may be used for ameliorating ortreating neuroblastoma. Thus, the present invention also providesmethods for ameliorating or treating neuroblastoma, comprisingadministering to a system or a subject in need thereof an effectiveamount of a compound having formula 1-5 or a pharmaceutical compositionthereof and optionally with an antiproliferative agent, whereby saidneuroblastoma is ameliorated or treated.

The present invention also provides methods for ameliorating or treatinga condition comprising administering to a subject or a system in needthereof any compound having formula 1-5 or a pharmaceutical compositionthereof, wherein said compound may be dibenzyltrisulfide,di(p-chlorobenzyl)trisulfide, (p-chlorobenzyl)benzyltrisulfide,di(p-nitrobenzyl)trisulfide, di(3-phenyl-2-propenyl)-trisulfide,diphenyltrisulfide, or di(p-t-butylphenyl)trisulfide. The subject may bea human or an animal such as a mammal. The system may be a cell ortissue, or other systems where compounds may be administered in vitro.

In one embodiment, the present invention provides methods for treatingor ameliorating a cell proliferative disorder other than neuroblastoma,comprising administering to a system or a subject in need thereof aneffective amount of any compound having formula 1-5 or a pharmaceuticalcomposition thereof and optionally with an antiproliferative agent,whereby said cell proliferative disorder in said system or subject isameliorated or treated. The present invention also provides methods forreducing or inhibiting cell proliferation or for inducing cell death.The present invention further provides methods for inducing apoptosis.In particular examples, the compound used in the methods of the presentinvention is dibenzyltrisulfide, di(p-fluorobenzyl)trisulfide,di(p-methylbenzyl)trisulfide or di(m-methylbenzyl)trisulfide, andoptionally with an antiproliferative agent.

In one aspect, cell proliferation is reduced, or said cell death isinduced. The cell proliferative disorder may be a tumor or a cancerincluding but not limited to leukemia, lymphoma, lung cancer, coloncancer, CNS cancer, melanoma, ovarian cancer, renal cancer, prostatecancer, breast cancer, head-neck cancer, pancreatic cancer, or renalcancer. In another aspect, cell apoptosis is induced. In another aspect,tubulin assembly or disassembly is disrupted, or G2/M progression of thecell cycle, cell mitosis, or a combination thereof, is inhibited. In yetanother aspect, endothelial cell proliferation, angiogenesis, or acombination thereof, is inhibited.

In another embodiment, the present invention provides methods forameliorating or treating restenosis, comprising administering to asubject in need thereof an effective amount of any compound havingformula 1-5 or a pharmaceutical composition thereof, whereby restenosisin said subject is ameliorated or treated. The restenosis may beassociated with neointimal hyperplasia. The compounds may beadministered via oral or parental administration, or via a stent. In yetanother embodiment, the present invention provides a pharmaceuticalcomposition for the treatment of a cell proliferative disorder,comprising any compound having formula 1-5, and a pharmaceuticallyacceptable excipient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C show the responses of H460 cells (non-small cell lung cancerline) to different concentrations of DBTS, colcemid, and paclitaxel,respectively, as determined on Real-Time Electronic Sensing System(RT-CES system).

FIG. 2 shows the responses of MV522 cells (lung cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 3 shows responses of MCF-7 cells (breast cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 4 shows responses of A549 cells (lung cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 5 shows responses of PC3 cells (prostate cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS) (FIG. 6A) and5-fluorouracil (FIG. 6B), as determined on RT-CES system.

FIGS. 6A and 6B shows responses of A431 cells (epidermoid cancer cellline) to different concentrations of dibenzyl trisulfide (DBTS), asdetermined on RT-CES system.

FIG. 7 shows responses of HT1080 cells (fibrosarcoma cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 8 shows responses of MDA-231 cells (breast cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 9 shows responses of HT-29 cells (colon cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 10 shows responses of HC-2998 cells (colon cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 11 shows responses of OVCAR4 cells (ovarian cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 12 shows responses of A2780 cells (colon cancer cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 13 shows responses of HepG2 cells (human hepatoma cell line) todifferent concentrations of dibenzyl trisulfide (DBTS), as determined onRT-CES system.

FIG. 14 shows mouse sarcoma S180 tumors (planted into mice bysubcutaneous implanting) treated with dibenzyl trisulfide (DBTS).

FIG. 15 shows mouse Lewis lung cancer (planted into mice by subcutaneousimplanting) treated with dibenzyl trisulfide (DBTS).

FIG. 16 shows Bcap-37 human breast tumors that werexenograft-transplanted in immunodeficient nude mice by subcutaneousseeding and were treated with compound ACEA100108.

FIG. 17 shows the dynamic change in tumor size in the in vivo antitumorefficacy test of compound ACEA100108 on Bcap-37 human breast cancer thatwas xenograft transplanted in immunodeficient nude mice by subcutaneousimplanting.

FIG. 18 shows the dynamic change in body weight of carrier mice in thein vivo antitumor efficacy test of compound ACEA100108 (100108) onBcap-37 human breast cancer that was xenograft-transplanted inimmunodeficient nude mice by subcutaneous implanting.

FIG. 19 shows HCT-8 human colon tumors that were xenograft-transplantedin immunodeficient nude mice by subcutaneous seeding and were treatedwith compound ACEA100108.

FIG. 20 shows the dynamic change in tumor size in the in vivo antitumorefficacy test of compound ACEA100108 on HCT-8 human colon cancer thatwas xenograft-transplanted in immunodeficient nude mice by subcutaneousimplanting.

FIG. 21 shows the dynamic change in body weight of carrier mice in thein vivo antitumor efficacy test of compound ACEA100108 (100108) on HCT-8human colon cancer that was xenograft-transplanted in immunodeficientnude mice by subcutaneous implanting.

FIG. 22 shows ao10/17 human ovarian tumors that werexenograft-transplanted in immunodeficient nude mice by subcutaneousseeding and were treated with compound ACEA100108.

FIG. 23 shows the dynamic change in tumor size in the in vivo antitumorefficacy test of compound ACEA100108 on ao10/17 human ovarian cancerthat was xenograft transplanted in immunodeficient nude mice bysubcutaneous implanting.

FIG. 24 shows the dynamic change in body weight of carrier mice in thein vivo antitumor efficacy test of compound ACEA100108 (100108) onao10/17 human ovarian cancer that was xenograft-transplanted inimmunodeficient nude mice by subcutaneous implanting.

FIG. 25 shows Bcap-37 human breast tumors that werexenograft-transplanted in immunodeficient nude mice by subcutaneousimplanting and were treated with compound ACEA100108.

FIG. 26 shows the responses of various cell lines to ACEA100108, asdetermined on RT-CES system.

FIG. 27 shows the responses of HT1080 cell to different derivatives ofDBTS, as determined on RT-CES system.

FIG. 28 shows the images of microtubules in control COS cells that werenot treated with any drugs.

FIG. 29 shows the images of microtubules in COS cells treated withdifferent concentrations of paclitaxel for 4 hours.

FIG. 30 shows the images of microtubules in COS cells treated withdifferent concentrations of paclitaxel for 24 hours.

FIG. 31 shows the images of microtubules in COS cells treated withdifferent concentrations of vinblastine for 4 hours.

FIG. 32 shows the images of microtubules in COS cells treated withdifferent concentrations of vinblastine for 24 hours.

FIG. 33 shows the images of microtubules in COS cells treated withdifferent concentrations of DBTS for 4 hours.

FIG. 34 shows the images of microtubules in COS cells treated withdifferent concentrations of DBTS for 24 hours.

FIG. 35 shows the images of microtubules in COS cells treated withdifferent concentrations of ACEA100108 for 4 hours.

FIG. 36 shows the images of microtubules in COS cells treated withdifferent concentrations of ACEA100108 for 24 hours.

FIG. 37 shows the images of microtubules in COS cells treated withdifferent concentrations of ACEA100116 for 4 hours.

FIG. 38 shows the images of microtubules in COS cells treated withdifferent concentrations of ACEA100116 for 24 hours.

FIG. 39 a shows the result of the in vitro microtubule assembly assaysusing pure tubulin (MAP-free) and DBTS.

FIG. 39 b shows the electron microscopic images of microtubulesassembled in vitro in the absence of any drug.

FIG. 39 c shows the electron microscopic images of microtubulesassembled in vitro in the presence of 3 uM DBTS.

FIG. 40 shows the result of the in vitro microtubule assembly assaysusing pure tubulin (MAP-free) and ACEA100108.

FIG. 41 shows the result of the in vitro microtubule assembly assaysusing pure tubulin (MAP-free) and ACEA100116.

FIG. 42 shows the fluorescent microscope images of 6-CFDA (top panel)and Annexin V (bottom panel) staining of A549 human lung cancer cellstreated with treated with 1 uM ACEA100108, 50 nM paclitaxel, 10 nMvinblastine or DMSO for 24 hrs.

FIG. 43 show the cell cycle distribution of A549 human lung cancer cellsafter they were treated with 25 uM ACEA100108, 7.8 nM paclitaxel, orDMSO for 24 hrs, as analyzed on a flow cytometry.

MODES OF CARRYING OUT THE INVENTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into the subsections thatfollow.

A. Definition

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications referred to herein areincorporated by reference in their entirety. If a definition set forthin this section is contrary to or otherwise inconsistent with adefinition set forth in the patents, applications, publishedapplications and other publications that are herein incorporated byreference, the definition set forth in this section prevails over thedefinition incorporated herein by reference.

As used herein, “a” or “an” means “at least one” or “one or more”.

The term “alkyl” as used herein refers to saturated hydrocarbon groupsin a straight, branched, or cyclic configuration and particularlycontemplated alkyl groups include lower alkyl groups (i.e., those havingten or less carbon atoms). Exemplary alkyl groups are methyl, ethyl,propyl, isopropyl, butyl, sec-butyl, tertiary butyl, pentyl, isopentyl,hexyl, etc. The term “alkenyl” as used herein refers to an alkyl asdefined above and having at least one double bond. Thus, particularlycontemplated alkenyl groups include straight, branched, or cyclicalkenyl groups having two to ten carbon atoms (e.g., ethenyl, propenyl,butenyl, pentenyl, etc.). Similarly, the term “alkynyl” as used hereinrefers to an alkyl or alkenyl as defined above and having at least onetriple bond. Especially contemplated alkynyls include straight,branched, or cyclic alkynes having two to ten total carbon atoms (e.g.,ethynyl, propynyl, butynyl, etc.).

The term “cycloalkyl” as used herein refers to a cyclic alkane (i.e., inwhich a chain of carbon atoms of a hydrocarbon forms a ring), preferablyincluding three to eight carbon atoms. Thus, exemplary cycloalkanesinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,and cyclooctyl. Cycloalkyls also include one or two double bonds, whichform the “cycloalkenyl” groups. Cycloalkyl groups are also furthersubstituted by alkyl, alkenyl, alkynyl, halo and other general groups.

The term “aryl” or “aromatic moiety” as used herein refers to anaromatic ring system, which may further include one or more non-carbonatoms. Thus, contemplated aryl groups include (e.g., phenyl, naphthyl,etc.) and pyridyl. Further contemplated aryl groups may be fused (i.e.,covalently bound with 2 atoms on the first aromatic ring) with one ortwo 5- or 6-membered aryl or heterocyclic group, and are thus termed“fused aryl” or “fused aromatic”.

As also used herein, the terms “heterocycle”, “cycloheteroalkyl”, and“heterocyclic moieties” are used interchangeably herein and refer to anycompound in which a plurality of atoms form a ring via a plurality ofcovalent bonds, wherein the ring includes at least one atom other than acarbon atom. Particularly contemplated heterocyclic bases include 5- and6-membered rings with nitrogen, sulfur, or oxygen as the non-carbon atom(e.g., imidazole, pyrrole, triazole, dihydro pyrimidine, indole,pyridine, thiazole, tetrazole etc.). Further contemplated heterocyclesmay be fused (i.e., covalently bound with two atoms on the firstheterocyclic ring) to one or two ring or heterocycle, and are thustermed “fused heterocycle” or “fused heterocyclic base” or “fusedheterocyclic moieties” as used herein.

The term “alkoxy” as used herein refers to straight or branched alkylconnecting to an oxygen atom called alkoxides, wherein the hydrocarbonportion may have any number of carbon atoms, may further include adouble or triple bond and may include one or two oxygen, sulfur ornitrogen atoms in the alkyl chains. For example, suitable alkoxy groupsinclude methoxy, ethoxy, propyloxy, isopropoxy, methoxyethoxy, etc.Similarly, the term “alkylthio” refers to straight or branched chainalkylsulfides, wherein the hydrocarbon portion may have any number ofcarbon atoms, may further include a double or triple bond and mayinclude one or two oxygen, sulfur or nitrogen atoms in the alkyl chains.For example, contemplated alkylthio groups include methylthio,ethylthio, isopropylthio, methoxyethylthio, etc.

Likewise, the term “alkylamino” refers to straight or branchedalkylamines, wherein the amino nitrogen “N” can be substituted by one ortwo alkyls and the hydrocarbon portion may have any number of carbonatoms and may further include a double or triple bond. Furthermore, thehydrogen of the alkylamino may be substituted with another alkyl group.Therefore, exemplary alkylamino groups include methylamino,dimethylamino, ethylamino, diethylamino, etc.

The term “aryloxy” as used herein refers to an aryl group connecting toan oxygen atom, wherein the aryl group may be further substituted. Forexample suitable aryloxy groups include phenyloxy, etc. Similarly, theterm “arylthio” as used herein refers to an aryl group connecting to asulfur atom, wherein the aryl group may be further substituted. Forexample suitable arylthio groups include phenylthio, etc.

The term “halogen” as used herein refers to fluorine, chlorine, bromineand iodine.

The term “amino acid” as used herein refers to substituted natural andunnatural amino acid with D- or L-configuration or the mixture in whichamino and acid groups are used to derivatize the contemplated compounds.

It should further be recognized that all of the above-defined groups mayfurther be substituted with one or more substituents, which may in turnbe substituted as well. For example, an “alkyl” as used hereinencompasses alkyls substituted with a heteroatom.

The term “substituted” as used herein refers to a replacement of an atomor chemical group (e.g., H, NH₂, or OH) with a functional group, andparticularly contemplated functional groups include nucleophilic groups(e.g., —NH₂, —OH, —SH, —NC, etc.), electrophilic groups (e.g., C(O)OR,C(X) OH, etc.), polar groups (e.g., —OH), non-polar groups (e.g.,heterocycle, aryl, alkyl, alkenyl, alkynyl, etc.), ionic groups (e.g.,—NH₃ ⁺), and halogens (e.g., —F, —Cl), NHCOR, NHCONH₂, OCH₂COOH,OCH₂CONH₂, OCH₂CONHR, NHCH₂COOH, NHCH₂CONH₂, NHSO₂R, OCH₂-heterocycles,PO₃H, SO₃H, amino acids, and various combinations known in the art.Moreover, the term “substituted” also includes multiple degrees ofsubstitution, and where multiple substituents are disclosed or claimed,the substituted compound can be independently substituted by one or moreof the disclosed or claimed substituent moieties.

The term “organo sulfur derivative” as used herein refers to an organiccompound containing two or more “S” atoms. The term “disulfide”,“trisulfide”, “tetrasulfide” or pentasulfide” as used herein refers to amoiety where two, three, four, or five sulfur atoms connect in a linearchain (—S—S—S—), where one or two or three of them may be furtheroxidized into S═O or SO₂, and where the di-, tri-, tetra- andpenta-sulfide derivatives are substituted with two functional, aryl,alkenyl, heterocyclic groups or substituents at the two ends of the di-,tri-, tetra- and penta-sulfide (R—S—(S)₀₋₃—S—R). Two or more trisulfide(—S—S—S—) moieties may be connected together by an aromatic or linearchain, which also refers to “trisulfide” or organo sulfide. One or twotrisulfide or organo sulfide moieties may be connected together to formcyclic ring systems.

B. Substituted Organo Sulfur Derivatives and Pharmaceutical CompositionsThereof

The present invention compounds having formula

wherein A and B are the same or different, and are independently anoptionally substituted aryl, heteroaryl, or a 5-14 membered ring whichmay be monocyclic or multicyclic and optionally containing a heteroatom;

each S is optionally in the form of an oxide;

S¹ and S2 are independently S, SO or SO₂;

each R is H, halogen, carboxyl, cyano, amino, amido, an amino acid, aninorganic substituent, SR¹, OR¹ or R¹, wherein each R¹ is alkyl,alkenyl, alkynyl, aryl, heteroaryl, a carbocyclic ring or a heterocyclicring, each of which is optionally substituted and may contain aheteroatom;

m, n and p are independently 0-3;

or a compound having formula (3) or (4):

wherein A, B, R, S, n and p are as defined above;

or a compound having formula (5):

wherein A, B, S, n and p are as defined above; and

Z is (CR¹ ₂)_(q) or (CR¹═CR¹)_(q)* wherein q is 0-3 and the * representsthat C═C may be replaced with alkynyl, O, S, NR; or Z is an optionallysubstituted aryl, heteroaryl or heterocyclic ring;

wherein A and B together may form a cyclic ring system;

and a pharmaceutically acceptable salt, ester, prodrug or metabolitethereof;

provided said compound is not dibenzyltrisulfide,di(p-chlorobenzyl)trisulfide, (p-chlorobenzyl)benzyltrisulfide,di(p-nitrobenzyl)trisulfide, di(3-phenyl-2-propenyl)-trisulfide,diphenyltrisulfide, or di(p-t-butylphenyl)trisulfide.

In other embodiments, each R in the above formula 1-5 may be anon-interfering substituent. In general, a “noninterfering substituent”is a substituent whose presence does not destroy the ability of acompound to behave as a therapeutic agent. For example, anon-interfering substituent may improve potency and PK properties. Inanother example, the non-interfering substituent may reduce toxicity.Suitable noninterfering substituents include halo, nitro, carboxyl,alkyl, alkenyl, alkynyl, aryl, arylalkyl, arylalkenyl, alkoxy,alkylthio, arylalkynyl, heterocycles, amino acids, each of which mayfurther be substituted with one or more non-interfering substituents.Noninterfering substituents may also include COOR, SR, OR, wherein R isalso a non-interfering substituent, as defined above.

In the above formula 1-5, A and B may independently be

where X and W are independently S, O, NR₇, CR₇;

or one W in a 6-membered monocyclic or bicyclic ring may be a bond; and

each R₁, R₂, R₃, R₄, R₅, R₆, R₇ is as previously defined.

In other embodiments, each R₁, R₂, R₃, R₄, R₅, R₆, R₇ may be a polar ornon-polar substituent. In other examples, each R₁, R₂, R₃, R₄, R₅, R₆,R₇ may be a nucleophilic or electrophilic non-interfering substituent.

The present invention also encompasses compounds having formula 1-5, aswell as their salts and prodrugs. Such salts, for example, may be formedfrom a positively charged substitute group (e.g. an amino group on Aand/or B) on a compound and a pharmaceutically suitable anion. Suitableanions include, but not limited to, chloride, bromide, iodide, sulfate,nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,maleate, and acetate. Pharmaceutically acceptable salts may also beformed from a negatively charged substituted group (e.g., carboxylategroup on A and/or B) on a compound and a cation. Non-limiting examplesof suitable cations are sodium ion, potassium ion, magnesium ion,calcium ion, and a organic ammonium ion such as tetramethylammonium ion,tetrabutylammonium ion, and other organic cations.

The trisulfides may be synthesized following procedures as illustratedin Scheme 1. For example, the aromatic or heterocyclic methylene halides(X═I or Br or Cl) are reacted with thiourea. The resulted isothiouroniumhalides are treated with sodium hydroxide to provide the correspondingthiol derivatives (Furniss, B. S.; Hannaford, A. J.; Rogers, V.; Smith,P. W. G.; Tatchell, A. R. Vogel's Textbook of Practical OrganicChemistry, Longman Group Limited, London, 1978, pp 582-583).

The symmetric trisulfide derivatives may be synthesized using Method A.In Method A, N-trimethylsilylimidazole is reacted with sulfurdichloride. The resulting di-imidazolylsulfide is then reacted withthiol to give the corresponding trisulfides. Method B can be used tosynthesize symmetric and asymmetric trisulfides. In Method B, the firstthiol is reacted with sulfur dichloride quantitatively at lowtemperature. The resulting intermediate thiosulfenyl chloride is thenreacted with the second thiol to provide the desired asymmetric orsymmetric trisulfide, depending on the thiol used in the second step.

The representative aromatic methylene thiols 1-6 (Scheme 2) may besynthesized using the similar procedure as described in Vogel'sPractical Organic Chemistry, pp 582-583. In addition, symmetrictrisulfide derivatives 7-32 (Scheme 2) were synthesized by Method Asimilar to the reported procedure (Banerji, A.; Kalena, G. P.Tetrahedron Letters 1980, 21, 3003-3004). For example, sulfur dichloride(14 mmol) in anhydrous hexanes or dichloromethane was added to a stirredsolution of N-trimethylsilylimidazole (28 mmol) in hexanes at roomtemperature. After stirring for 30 minutes, the reaction mixture wascooled to 0° C., and a solution of designated thiol (28 mmol) inanhydrous hexanes was added dropwise for a period of 30 minutes. Thereaction mixture was stirred for 30 minutes, and the precipitatedimidazole by-product was filtered off. The filtrate was washed withwater and brine, and dried over anhydrous sodium sulfate. The solventwas evaporated, and the residue is purified by flash chromatography on asilica gel column using hexanes-ethyl acetate 100:1 to 20:1 as eluentsto provide desired trisulfides 7-32 in 60-90% yields. The aromatictrisulfides 33-39 were synthesized by the similar procedure in 30-70%yields.

Di(p-fluorobenzyl)trisulfide (8). Trisulfide 8 was synthesized in 77%yield. The white crystalline was obtained by chromatographicpurification followed by recrystallization from hexanes. Silica gel TLCR_(f)=0.46 (40:1 hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ4.00 (s, 4H), 7.01 (t, 4H, J=8.8 Hz), 7.27 (dd, 4H, J=8.8, 5.4 Hz); ¹³CNMR (125.7 MHz, CDCl₃) δ 42.4, 115.6, 115.8, 131.2, 131.3, 132.4, 162.5(C—F, J=250 Hz); ¹⁹F NMR (376.5 MHz, CDCl₃) δ −114.2; ES MS m/z 337/338(M+Na)⁺; Anal. Calcd. for C₁₄H₁₂F₂S₃: C, 53.48; H, 3.85; S, 30.59.Found: C, 53.16; H, 4.22; S, 30.24.

Di(p-chlorobenzyl)trisulfide (9). Trisulfide 9 was synthesized in 90%yield. The white crystalline was obtained by chromatographicpurification followed by recrystallization from hexanes. Silica gel TLCR_(f)=0.45 (40:1 hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ3.98 (s, 4H), 7.22 (d, 4H, J=8.4 Hz), 7.29 (d, 4H, J=8.4 Hz).

Di(m-trifluoromethylbenzyl)trisulfide (12). Trisulfide 12 wassynthesized in 99% yield. The white crystalline was obtained bychromatographic purification followed by recrystallization from hexanes.Silica gel TLC R_(f)=0.33 (40:1 hexanes-ethyl acetate). ¹H NMR (499.1MHz, CDCl₃) δ 4.04 (s, 4H), 7.41-7.49 (m, 4H), 7.51-7.58 (m, 4H).

Di(benzo[B]thiophen-3-yl-methane)trisulfide (22). Trisulfide 22 wassynthesized in 45% yield. The white solid was obtained bychromatographic purification. Silica gel TLC R_(f)=0.45 (40:1hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ 3.74 (s, 4H), 7.01(s, 2H), 7.34-7.45 (m, 4H), 7.75 (d, 2H, J=7.4 Hz), 7.85 (dd, 2H, J=7.8,1.1 Hz). ES MS m/z 391 (M+H)⁺, 413 (M+Na)⁺.

Di(p-bromobenzyl)trisulfide (25). Trisulfide 25 was synthesized in 84%yield. The white crystalline was obtained by chromatographicpurification followed by recrystallization from hexanes. Silica gel TLCR_(f)=0.55 (40:1 hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ3.96 (s, 4H), 7.17 (d, 4H, J=8.3 Hz), 7.45 (d, 4H, J=8.3 Hz).

Di(p-methylbenzyl)trisulfide (26). Trisulfide 26 was synthesized in 99%yield. The white crystalline was obtained by chromatographicpurification followed by recrystallization from hexanes. Silica gel TLCR_(f)=0.66 (40:1 hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ2.33 (s, 6H), 4.01 (s, 4H), 7.14 (d, 4H, J=8.0 Hz), 7.21 (d, 4H, J=8.0Hz).

Di(p-t-butylbenzyl)trisulfide (28). Trisulfide 28 was synthesized in 96%yield. The white crystalline was obtained by chromatographicpurification followed by recrystallization from hexanes. Silica gel TLCR_(f)=0.50 (40:1 hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ1.30 (s, 18H), 4.02 (s, 4H), 7.25 (d, 4H, J=8.3 Hz), 7.35 (d, 4H, J=8.3Hz).

Di(o-chlorobenzyl)trisulfide (30). Trisulfide 30 was synthesized in 77%yield. The white crystalline was obtained by chromatographicpurification followed by recrystallization from hexanes. Silica gel TLCR_(f)=0.44 (40:1 hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ4.17 (s, 4H), 7.23-7.28 (m, 4H), 7.35-7.43 (m, 4H).

Di(2,4,6-trimethylbenzyl)trisulfide (32). Trisulfide 32 was synthesizedin 59% yield. The white crystalline was obtained by chromatographicpurification followed by recrystallization from hexanes. Silica gel TLCR_(f)=0.65 (40:1 hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ2.27 (s, 6H), 2.42 (s, 12H), 4.23 (s, 4H), 6.87 (s, 4H).

Di(p-methoxyphenyl)trisulfide (33). Trisulfide 33 was synthesized in 98%yield. The white crystalline was obtained by chromatographicpurification followed by recrystallization from hexanes. Silica gel TLCR_(f)=0.32 (20:1 hexanes-ethyl acetate). ¹H NMR (499.1 MHz, CDCl₃) δ3.80 (s, 4H), 6.81 (d, 4H, J=8.8 Hz), 7.47 (d, 4H, J=8.8 Hz).

Di(4-trifluoromethylpyridin-2-yl)trisulfide (34). Trisulfide 34 wassynthesized in 53% yield. The white crystalline was obtained bychromatographic purification followed by recrystallization from hexanes.Silica gel TLC R_(f)=0.61 (10:1 hexanes-ethyl acetate). ¹H NMR (499.1MHz, CDCl₃) δ 7.70 (d, 4H, J=8.4 Hz), 7.84 (dd, 4H, J=8.4, 2.4 Hz), 8.73(s, 2H).

The asymmetric trisulfide derivatives listed in Tables 1-8 may besynthesized following similar procedures as for compounds 41-68, usingthe corresponding thiol.

TABLE 1 Various disubstituted trisulfide derivatives.

TABLE 2 Various disubstituted trisulfide derivatives.

TABLE 3 Various disubstituted trisulfide derivatives.

TABLE 4 Various disubstituted trisulfide derivatives.

TABLE 5 Various disubstituted trisulfide derivatives.

TABLE 6 Various substituted trisulfide derivatives.

TABLE 7 Various disubstituted trisulfide derivatives.

TABLE 8 Various disubstituted trisulfide derivatives.

The di-substituted(trisulfide) derivatives listed in Schemes 4 and 5 maybe synthesized by similar procedures (Method B). For example, a solutionof 1,3-benzenedimethanethiol or 2-butene-1,4-dithiol (10 mmol) andanhydrous pyridine (20 mmol) in 30 mL of diethyl ether is added dropwiseover a period of 30 minutes to a cold (−78° C.) stirred solution ofsulfur dichloride (20 mmol) in 80 mL of anhydrous diethyl ether. Thereaction mixture is stirred for 30 minutes. The corresponding secondthiol (20 mmol) and anhydrous pyridine (20 mmol) in 40 mL of diethylether is added dropwise over a period of 30 minutes at −78° C., and thereaction mixture is further stirred for an additional 30 minutes. Thereaction mixture is washed with water (2 times), 1 N sodium hydroxidesolution (2 times), and then water (2 times) until pH is neutral. Theorganic phase is dried over CaCl₂ or anhydrous sodium sulfate, filteredand concentrated. The residue is passed through a short pad of silicagel using hexanes-ethyl acetate as eluent to provide di-substitutedtrisulfides in 40-90% yields.

The trisulfide derivatives may be synthesized by the methods describedabove or by the approach illustrated in Scheme 6. The tetra- andpenta-sulfide derivatives are synthesized by the similar strategy basedon the reported procedure (Sinha, P.; Jundu, A.; Roy, S.; Prabhakar, S.;Vairamani, M.; Sankar, A. R.; Kunwar, A. C. Organometallics 2001, 20,157-162).

The symmetric or asymmetric sulfenic sulfonic thioanhydride derivatives(Scheme 7) can be synthesized based on the reported procedures (Karpp,D. N.; Gleason, J. G.; Ash, D. K. J. Org. Chem. 1971, 36, 322-326; andHarpp, D. N.; Ash, D. K.; Smith, R. A. J. Org. Chem. 1979, 44,4135-4140).

The present invention also provides pharmaceutical compositionscomprising an effective amount of a compound having formula 1-5optionally with an antiproliferative agent, and a pharmaceuticallyacceptable excipient. As used herein, an “effective amount” refers tothe amount of the compound which is required to confer a therapeuticeffect on the treated subject. The effective amount or dose will vary asrecognized by those skilled in the art, depending on the types of tumorstreated, route of administration, and possible co-administration withother therapeutic treatments such as use of other anti-tumor agents orradiation therapy.

As used herein, the term “antiproliferative agent” refers to atherapeutic agent that may be used for treating or ameliorating a cellproliferative disorder such as tumors or cancer. Examples ofantiproliferative agents include but are not limited to anantineoplastic agent, an alkylating agent, a plant alkaloid, anantimicrobial agent, a sulfonamide, an antiviral agent, a platinumagent, and other anticancer agents known in the art. Particular examplesof antiproliferative agents include but are not limited to cisplatin,carboplatin, busulphan, methotrexate, daunorubicin, doxorubicin,cyclophosphamide, mephalan, vincristine, vinblastine, chlorambucil,paclitaxel, gemcitabine, and others known in the art. (See e.g., Goodman& Gilman's, The Pharmacological Basis of Therapeutics (9th Ed) (Goodman,et al., eds.) (McGraw-Hill) (1996); and 1999 Physician's Desk Reference(1998)).

Any suitable formulation of the compounds described herein may beprepared. In cases where compounds are sufficiently basic or acidic toform stable nontoxic acid or base salts, administration of the compoundsas salts may be appropriate. Examples of pharmaceutically acceptablesalts are organic acid addition salts formed with acids that form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate,α-ketoglutarate, and α-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts. Pharmaceutically acceptable salts are obtainedusing standard procedures well known in the art, for example, by asufficiently basic compound such as an amine with a suitable acid,affording a physiologically acceptable anion. Alkali metal (e.g.,sodium, potassium or lithium) or alkaline earth metal (e.g., calcium)salts of carboxylic acids also are made.

The compounds having formula 1-5 as described herein are generallysoluble in organic solvents such as chloroform, dichloromethane, ethylacetate, ethanol, methanol, isopropanol, acetonitrile, glycerol,N,N-dimethylformamide, N,N-dimetheylaceatmide, dimethylsulfoxide, etc.In one embodiment, the present invention provides formulations preparedby admixing a compound having formula 1-5 with a pharmaceuticallyacceptable carrier. In one aspect, the formulation may be prepared usinga method comprising: a) dissolving a compound of claim 1 in awater-soluble organic solvent, a non-ionic solvent, a water-solublelipid, a cyclodextrin, a vitamin such as tocopherol, a fatty acid, afatty acid ester, a phospholipid, or a combination thereof, to provide asolution; and b) adding saline our a buffer containing 1-10%carbohydrate solution. In one example, the carbohydrate comprisesdextrose. The pharmaceutical compositions obtained using the presentmethods are stable and useful for animal and clinical applications.

Illustrative examples of water soluble organic solvents for use in thepresent methods include and are not limited to polyethylene glycol(PEG), alcohols, acetonitrile, N-methyl-2-pyrrolidone,N,N-dimethylformamide, N,N-dimethylacetamide, dimethyl sulfoxide, or acombination thereof. Examples of alcohols include but are not limited tomethanol, ethanol, isopropanol, glycerol, or propylene glycol.

Illustrative examples of water soluble non-ionic surfactants for use inthe present methods include but are not limited topolyoxyethyleneglycerol-triricinoleat 35, PEG-succinate, polysorbate 20,polysorbate 80, polyethylene glycol 660 12-hydroxystearate, sorbitanmonooleate, poloxamer, ethoxylated persic oil, capryl-caproylmacrogol-8-glyceride, glycerol ester, PEG 6 caprylic glyceride,glycerin, glycol-polysorbate, or a combination thereof. Particularexamples of non-ionic surfactants are polyethylene glycol modifiedCREMOPHOR® (polyoxyethyleneglyceroltriricinoleat 35), CREMOPHOR® EL,hydrogenated CREMOPHOR® RH40, hydrogenated CREMOPHOR® RH60, SOLUTOL® HS(polyethylene glycol 660 12-hydroxystearate), LABRAFIL® (ethoxylatedpersic oil), LABRASOL® (capryl-caproyl macrogol-8-glyceride), GELUCIRE®(glycerol ester), and SOFTIGEN® (PEG 6 caprylic glyceride).

Illustrative examples of water soluble lipids for use in the presentmethods include but are not limited to vegetable oils, triglycerides,plant oils, or a combination thereof. Examples of lipid oils include butare not limited to castor oil, polyoxyl castor oil, corn oil, olive oil,cottonseed oil, peanut oil, peppermint oil, safflower oil, sesame oil,soybean oil, hydrogenated vegetable oil, hydrogenated soybean oil, atriglyceride of coconut oil, palm seed oil, and hydrogenated formsthereof, or a combination thereof.

Illustrative examples of fatty acids and fatty acid esters for use inthe present methods include but are not limited to oleic acid,monoglycerides, diglycerides, a mono- or di-fatty acid ester of PEG, ora combination thereof.

Illustrative examples of cyclodextrins for use in the present methodsinclude but are not limited to alpha-cyclodextrin, beta-cyclodextrin,hydroxypropyl-beta-cyclodextrin, or sulfobutyl ether-beta-cyclodextrin.

Illustrative examples of phospholipids for use in the present methodsinclude but are not limited to soy phosphatidylcholine, or distearoylphosphatidylglycerol, and hydrogenated forms thereof, or a combinationthereof.

One of ordinary skill in the art may modify the formulations within theteachings of the specification to provide numerous formulations for aparticular route of administration. In particular, the compounds may bemodified to render them more soluble in water or other vehicle. It isalso well within the ordinary skill of the art to modify the route ofadministration and dosage regimen of a particular compound in order tomanage the pharmacokinetics of the present compounds for maximumbeneficial effect in a patient.

C. Methods of Using Substituted Organo Sulfur Derivatives andPharmaceutical Compositions Thereof

The compounds as described herein may be used as cytotoxic and/orcytostatic agents in treating cancers or other types of proliferativedisease. These compounds may function through any type of actionmechanisms. For example, the compounds may inhibit G2/M progression ofthe cell cycle, which might eventually induce apoptosis in tumor cells(see, e.g., Weung, et al. Biochim. Biophys. Res. Comm. 1997, 263,398-404). Some compounds may disrupt tubulin assembly, and othercompounds may disrupt tubulin disassembly, which may inhibit cellmitosis and induce cell apoptosis (see, e.g., Panda, et al. Proc. Natl.Acad. Sci. USA, 1997, 94, 10560-10564). The compounds may also inhibitendothelial cell proliferation and angiogenesis effect (see, e.g.,Witte, et al. Cancer Metastasis Rev. 1998, 17, 155-161).

The present invention also provides pharmaceutical compositions for thetreatment of a cell proliferative disorder, comprising any compoundhaving formula 1-5, including but not limited to dibenzyltrisulfide,di(p-chlorobenzyl)trisulfide, (p-chlorobenzyl)benzyltrisulfide,di(p-nitrobenzyl)trisulfide, di(3-phenyl-2-propenyl)-trisulfide,diphenyltrisulfide, or di(p-t-butylphenyl)trisulfide.

To practice the method of the present invention, compounds havingformula 1-5 and pharmaceutical compositions thereof may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally, via an implanted reservoir, or other drugadministration methods. The term “parenteral” as used herein includessubcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional and intracranial injection or infusion techniques.

A sterile injectable composition, such as a sterile injectable aqueousor oleaginous suspension, may be formulated according to techniquesknown in the art using suitable dispersing or wetting agents andsuspending agents. The sterile injectable preparation may also be asterile injectable solution or suspension in a non-toxic parenterallyacceptable diluent or solvent. Among the acceptable vehicles andsolvents that may be employed include mannitol, water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium (e.g.,synthetic mono- or diglycerides). Fatty acids, such as oleic acid andits glyceride derivatives, are useful in the preparation of injectables,as are pharmaceutically acceptable oils, such as olive oil or castoroil, especially in their polyoxyethylated versions. These oil solutionsor suspensions can also contain a long-chain alcohol diluent ordispersant, or carboxymethyl cellulose or similar dispersing agents.Various emulsifying agents or bioavailability enhancers which arecommonly used in the manufacture of pharmaceutically acceptable solid,liquid, or other dosage forms can also be used for the purpose offormulation.

A composition for oral administration may be any orally acceptabledosage form including, but not limited to, tablets, capsules, emulsionsand aqueous suspensions, dispersions and solutions. In the case oftablets for oral use, commonly used carriers include lactose and cornstarch. Lubricating agents, such as magnesium stearate, can also beadded. For oral administration in a capsule form, useful diluentsinclude lactose and dried corn starch. When aqueous suspensions oremulsions are administered orally, the active ingredient can besuspended or dissolved in an oily phase combined with emulsifying orsuspending agents. If needed, certain sweetening, flavoring, or coloringagents can be added. A nasal aerosol or inhalation compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation and can be prepared as solutions in, for example saline,employing suitable preservatives (for example, benzyl alcohol),absorption promoters to enhance bioavailability, and/or othersolubilizing or dispersing agents known in the art.

In addition, the compounds having formula 1-5 may be administered aloneor in combination with other anticancer agents for the treatment ofvarious cancers or conditions. Combination therapies according to thepresent invention comprise the administration of at least one compoundof the present invention or a functional derivative thereof and at leastone other pharmaceutically active ingredient. The active ingredient(s)and pharmaceutically active agents may be administered separately ortogether. The amounts of the active ingredient(s) and pharmaceuticallyactive agent(s) and the relative timings of administration will beselected in order to achieve the desired combined therapeutic effect.

In one embodiment, the present invention is directed to a method oftreating or ameliorating a cancer of a tissue or organ, including butnot limited to leukemia, lymphoma, lung cancer, colon cancer, CNScancer, melanoma, ovarian cancer, renal cancer, prostate cancer, breastcancer, pancreatic cancer, renal cancer, and other types ofproliferative disease comprising administering a therapeuticallyeffective amount of a compound having formula 1-5.

In another embodiment, the present invention is directed to a method oftreatment of restenosis after coronary stenting for patients withcoronary artery diseases with a compound having formula 1-5, such asdibenzyl trisulfide and other substituted trisulfide derivatives. One ofthe main causes of restenosis after coronary stenting for patients withcoronary artery disease is neointimal hyperplasia which may result fromthe proliferation and migration of smooth-muscle cells and extracellularmatrix productions (see, for example, “Pathology of acute and chroniccoronary stenting in humans”, by Farb, A., Sangiorgi, G., Certer, A. J.,et al. Circulation, 1999, 99, 44-52). Compounds that haveanti-proliferation capability may have an effect in reducing the risk ofclinical and angiographic restenosis when such compounds are deliveredwith a suitable means (see, for example, “A polymer-based,paclitaxel-eluting stent in patients with coronary artery disease”, byStone, G. W., Ellis, S. G., Cox, D. A, et al. New Engl. J. Med., 2004,350, 221-231). Thus, dibenzyl trisulfide and other compounds havingformula 1-5 may also be useful in inhibiting proliferation of the cellsinvolved in neointimal hyperplasia and thus reducing the incidence ofneointimal hyperplasia and restenosis.

Various methods may be used to effectively deliver compounds havingformula 1-5 to their target, such as cells. For example, a compositioncomprising dibenzyl trisulfide, or a another compound having formula 1-5may be administered orally, parenterally, or via an implanted reservoir.In other examples, the approaches described in the following papershereby incorporated by reference, may also be used: “A polymer-based,paclitaxel-eluting stent in patients with coronary artery disease”, byStone, G. W., Ellis, S. G., Cox, D. A. et al. New Engl. J. Med. 2004,350, 221-231; “A randomized comparison of a sirolimus-eluting stent witha standard stent for coronary revascularization”, by Morice, M.-C.,Serruys, P. W., Sousa, J. E., et al. New Engl. J. Med. 2002, 346,1773-1780; “Sirolimus-eluting stents versus standard stents in patientswith stenosis in a native coronary artery”, by Moses, J. W., Leon, M.B., Popma, J. J., et al, New Engl. J. Med. 2003, 349, 1315-1323.

The anticancer efficacy of dibenzyl trisulfide and substituted organosulfur analogues described above may be preliminarily screened in vitrousing a penal of cancer cell lines by standard endpoint assay formats(see below for the detailed description), or by real time electroniccell sensing (RT-CES) system, which provides dynamic cell responseinformation after exposing to an anticancer agent. Several endpointcell-based screening assay formats for anticancer agent discovery andvalidation may be used. For example, National Cancer Institute (NCI)provides an endpoint cytotoxicity assay system using a panel of 60cancer cell lines, which can be used for a large scale of cell-basedscreening of anticancer agents. (See, e.g., Monks, A., et al. J Natl.Cancer Inst. 1991, 83, 757-766; Alley, M. C., et al. Cancer Res. 1988,48, 589-601; Shoemaker, R. H., et al. Proc. Clin. Biol. Res. 1988, 276,265-286; and Stinson, et al. Proc. Am. Asso. Cancer Res. 1989, 30, 613).

In this screening method, cell suspension that is diluted to a desiredcell concentration is added into wells of a 96-well microtiter plate sothat each well is having solution about 100 microliters with cell numberbetween thousands (for example, 5000) and tens of thousands (forexample, 40,000). The number of cells added to individual wells dependson cell type, cell size, cell growth characteristics. Cells in the plateare incubated at 37° C., saturated humidity and 5% CO₂ atmosphere in astandard cell culture incubator for about 24 hrs. Compounds of interestare prepared into test solutions with serial diluted concentrations. Inone example, the dilution factor in the serial diluted solutions is10-fold (or 2-, 3-, 4-fold) and five (or six to ten) differentconcentrations with a ratio of highest concentration to lowestconcentration of 10,000. Other dilution factors and other variousconcentrations may also be used. Typically, the highest concentration ofthe test compound is 10⁻⁴ M. About 100 microliters of test solutions areadded into each well at 24 hours after initial cell seeding into wells.Test solutions of each compound concentration are added into at leasttwo wells for replicating purpose. The test compound may be dissolved inan organic solvent such as DMSO, and the 100 microliter test solutionsmay be a mixture of aqueous solution with the organic solvent-basedsolution or suspension.

After compound addition, cells are then incubated with the compound foradditional 48 hours at 37° C. in 5% CO₂ atmosphere and saturatedhumidity. The cells can then be assayed for their viable cell numbers byvarious assays, for example, the sulforhodamine B assay (as described byRubinstein, L. V., et al. J. Natl. Cancer Inst. 1990, 82, 1113-1118; andSkehan, P., et al. J. Natl. Cancer Inst. 1990, 82, 1107-1112). A platereader is then used to read the optical densities and an IC₅₀ value, theconcentration of drug that causes 50% growth inhibition, (or GI₅₀ valueto emphasize the correction for the cells counted at time zero), isderived based on the dose response curves. Thus, GI₅₀ values are used tomeasure the growth inhibitory power of the test compound. See Boyd, etal in Cytotoxic Anticancer Drugs: Models and Concepts for Drug Discoveryand Development; Vleriote, F. A., Corbett T. H., Baker L. H. (Eds.);Kluwer Academic: Hingham, Mass., 1992, pp 11-34.

In another assay format, a test compound is assayed for its cytotoxicityand/or cytostatic effect on certain cancer cell types, using endpointassay methods. Cells in the NCI cancer cell panel may be used. Cellsafter a pre-incubation for certain length of time (for example, 8 hrs or24 hrs) are incubated with a test compound at serially-dilutedconcentrations (for example, five 10-fold dilutions) for 24 hrs and/or48 hrs, and/or other specific length of time. The dose dependentcytotoxicity and/or cytostatic effects of test compounds can then betested and evaluated using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assaymethod, as described by, for example, Boyd (In Principle of Practice ofOncology, Devita, J. T., Hellman, S. and Rosenberg S. A. (Eds), 1989,Vol, 3, PPO Update, No. 10).

Another in vitro assay may be used to evaluate the effect of compoundsin arresting the cell cycle progression. More specifically, a testcompound is added to cells of certain cell lines in aconcentration-dependent manner. After cells are incubated for certainspecific length of time, cells are stained using propidium iodide andare used for flow cytometric assessment. The cell populations ofsub-G0/G1, G0/G1, S and G2/M phases are determined. All above in vitroassays are cell-based, single-time point (or multiple-time points usingmultiple plates) end-point assays.

Test compounds may also be screened using a novel in vitro cell-basedscreening assay system based on the electronic measurement ofcell-substrate or cell-electrode impedances. In contrast to all theendpoint assay systems, the cell-based screening assay system allows forreal time monitoring dynamic response of cancer cells to anticanceragents without labeling cells. This system can also be used for a largescale of in vitro cell-based high throughput screening of anticanceragents. The approach features in the integration of molecular and cellbiology with microelectronics and is based on the electronic detectionof biological assay process.

The details of this cell electronic sensing technology, called real-timecell electronic sensing (RT-CES™) and associated devices, systems andmethods of use are described in U.S. provisional application No.60/397,749, filed on Jul. 20, 2002; U.S. provisional application No.60/435,400, filed on Dec. 20, 2002; U.S. Provisional application60/469,572, filed on May 9, 2003, PCT application number PCT/US03/22557,filed on Jul. 18, 2003; PCT application number PCT/US03/22537, filed onJul. 18, 2003; PCT application number PCT/US04/37696, filed on Nov. 12,2004; PCT application number PCT/US05/04481, filed on Feb. 9, 2005; U.S.patent application Ser. No. 10/705,447, filed on Nov. 10, 2003; U.S.patent application Ser. No. 10/705,615, filed on Nov. 10, 2003; U.S.patent application Ser. No. 10/987,732, filed on Nov. 12, 2004; U.S.patent application Ser. No. 11/055,639, filed on Feb. 9, 2005, each ofwhich is incorporated by reference. Additional details of RT-CEStechnology is further disclosed in U.S. provisional application No.60/519,567, filed on Nov. 12, 2003, and U.S. provisional application No.60/542,927, filed on Feb. 9, 2004, U.S. provisional application No.60/548,713, filed on Feb. 27, 2004, U.S. provisional application No.60/598,608, filed on Aug. 4, 2004; U.S. provisional application No.60/598,609, filed on Aug. 4, 2004; U.S. provisional application No.60/613,749, filed on Sep. 27, 2004; U.S. provisional application No.60/613,872, filed on Sep. 27, 2004; U.S. provisional application No.60/614,601, filed on Sep. 29, 2004; U.S. provisional application No.60/630,071, filed on Nov. 22, 2004; U.S. provisional application No.60/630,131, filed on Nov. 22, 2004, each of which is incorporated hereinby reference.

For measurement of cell-substrate or cell-electrode impedance usingRT-CES technology, microelectrodes having appropriate geometries arefabricated onto the bottom surfaces of microtiter plate or similardevice, facing into the wells. Cells are introduced into the wells ofthe devices, and make contact to and attach to the electrode surfaces.The presence, absence or change of properties of cells affects theelectronic and ionic passage on the electrode sensor surfaces. Measuringthe impedance between or among electrodes provides important informationabout biological status of cells present on the sensors. When there arechanges to the biological status of the cells analogue, electronicreadout signals are measured automatically and in real time, and areconverted to digital signals for processing and analysis. In a RT-CESsystem, a cell index is automatically derived and provided based onmeasured electrode impedance values. The cell index obtained for a givenwell reflects: 1) how many cells are attached to the electrode surfacesin this well; 2) how well cells are attached to the electrode surfacesin this well. Thus, the more the cells of same type in similarphysiological conditions attach the electrode surfaces, the larger thecell index. And, the better the cells attach to the electrode surfaces(e.g., the cells spread-out more to have larger contact areas, or thecells attach tighter to electrode surfaces), the larger the cell index.

Through the use of the RT-CES system, dibenzyl trisulfide has been shownto inhibit proliferation of a variety of cancer types. Dibenzyltrisulfide has not previously been found using standard endpoint assays.Negative conclusions that dibenzyl trisulfide has no antiproliferationactivity were made by the previous researchers (“Discovery of novelinducers of cellular differentiation using HL-60 promyelocytic cells”,Mata-Greenwood, E., Ito, A., Westernburg, H., Cui, B., Mehta, R. G.,Kinghorn, A. D. and Pezzuto, J. M. Anticancer Res. 2001, 21, 1763-1770).

To evaluate the anticancer efficacy and to predict possible mechanismsof the anticancer action of the dibenzyl trisulfide, ten anticancercompounds were tested with known mechanisms of action side by side withdibenzyl trisulfide utilizing a panel of 12 cancer cell lines. Thetime-dependent, cell responsive patterns of dibenzyl trisulfide (atcertain concentrations) were somewhat similar to those of paclitaxel,vinblastine and colceimid (at certain concentrations). Thus, dibenzyltrisulfide may have mechanisms of anticancer action similar to those ofpaclitaxel, vinblastine, and colceimid. Dibenzyl trisulfide may act oncancer cells through other mechanisms of action, different from those ofpaclitaxel, vinblastine and colceimid. It is also possible that dibenzyltrisulfide act on cancer cells through multiple mechanisms of action,including the mechanism of action similar to those of pacliotaxel,vinblastine and colceimid.

In addition to the in vitro cell models and assay formats, anti-tumoractivity of compounds can be further assessed and evaluated by in vivoanimal models with transplanted cancer. Most in vivo models are mousemodels.

In Vitro Cell-Based Screening Using Real-Time Cell Electronic Sensing(RT-CES) System

The RT-CES system comprises three components, an electronic sensoranalyzer, a device station and 16× or 96× microtiter plate devices.Microelectrode sensor array was fabricated on glass slides withlithographical microfabrication methods and the electrode-containingslides are assembled to plastic trays to form electrode-containingwells. Each 16× (or 96×) microtiter plate device used in RT-CES systemcomprises up to 16 (or 96) such electrode-containing wells. The devicestation receives the 16× or 96× microtiter plate devices and is capableof electronically switching any one of the wells to the sensor analyzerfor impedance measurement. In operation, the devices with cells culturedin the wells are placed into a device station that is located inside anincubator. Electrical cables connect the device station to the sensoranalyzer. Under the RT-CES software control, the sensor analyzer canautomatically select wells to be measured and continuously conductimpedance measurements. The impedance data from the analyzer istransferred to a computer, analyzed and processed by the integratedsoftware.

Impedance measured between electrodes in an individual well depends onelectrode geometry, ionic concentration in the well and whether thereare cells attached to the electrodes. In the absence of the cells,electrode impedance is mainly determined by the ion environment both atthe electrode/solution interface and in the bulk solution. In thepresence of the cells, cells attached to the electrode sensor surfaceswill alter the local ionic environment at the electrode/solutioninterface, leading to an increase in the impedance. The more cells thereare on the electrodes, the larger the increase in cell-electrodeimpedance. Furthermore, the impedance change also depends on cellmorphology and the extent to which cells attach to the electrodes.

To quantify cell status based on the measured cell-electrode impedance,a parameter termed Cell Index is derived, according to

${CI} = {\max\limits_{{i = 1},\ldots \mspace{14mu},N}\left( {\frac{R_{cell}\left( f_{i} \right)}{R_{b}\left( f_{i} \right)} - 1} \right)}$

where R_(b)(f) and R_(cell)(f) are the frequency dependent electroderesistances (a component of impedance) without cells or with cellpresent, respectively. N is the number of the frequency points at whichthe impedance is measured. Thus, Cell Index is a quantitative measure ofthe status of the cells in an electrode-containing well. Under the samephysiological conditions, more cells attached on to the electrodes leadsto larger R_(cell)(f) value, leading to a larger value for Cell Index.Furthermore, for the same number of cells present in the well, a changein the cell status such as morphology will lead to a change in the CellIndex. For example, an increase in cell adhesion or cell spreading leadsto larger cell-electrode contact area which will lead to an increase inR_(cell)(f) and thus a larger value for Cell Index. The Cell Index mayalso be calculated using a formula different from the one describedhere. Other methods for calculating the Cell Index based on impedancemeasurement can be found in PCT application number PCT/US04/37696, finedon Nov. 12, 2004, PCT application number PCT/US05/04481, filed on Feb.9, 2005, U.S. patent application Ser. No. 10/987,732, filed on Nov. 12,2004, and U.S. patent application Ser. No. 11/055,639, filed on Feb. 9,2005.

Different types of human cancer cells, including NCI-H460 (non-smallcell lung cancer cells), MV522 SW (non-small cell lung cancer cells),MCF7 (breast cancer cells), A549 (non-small cell lung cancer cells), PC3(prostate cancer cells), A431 (epidermoid cancer cells), HT1080(fibrosarcoma cells), MDA.MB2321 (breast cancer cells), HT29 (coloncancer cells), HCC2998 (colon cancer cells), OVCAR4 (ovarian cancercells), A2780 (ovarian cancer cells) and HepG2 (human hepatosarcoma)with different numbers (4000 to 20,000 per well) were seeded into 16× or96× microtiter device and monitored by RT-CES™ system. The cells wereallowed to grow for about 24 hours prior to the addition of dibenzyltrisulfide dissolved in DMSO solution (final DMSO concentration: 0.2%;final dibenzyl trisulfide concentration: between 1.5625 μM and 100 μM).The cell-electrode impedance was continuously measured and thecorresponding, time dependent cell-index values were derived andrecorded.

FIGS. 1-5, 6A, and 7-12 show the time-dependent cell index for a numberof cell lines prior to and after addition of dibenzyl trisulfide atvarious concentrations. As shown in the Figures, dibenzyl trisulfideexhibited inhibitory effect on the proliferation of a number of cancercell lines. The susceptibility to dibenzyl trisulfide differs among thecancer cell types. For some cancer cell types, a low dosage of dibenzyltrisulfide is sufficient to significantly inhibit cancer cellproliferation, whilst for other cancer cell types, a higher dosage isneeded to achieve similar inhibition degree.

In one example, FIGS. 1B and 1C show the time-dependent cell index forH460 (non-small cell lung cancer cell line) cells prior to and afteraddition of colcemid and paclitaxel at various concentrations. As shownin FIGS. 1B and 1C, colcemid and paclitaxel exhibited inhibitory abilityagainst the proliferation of A431 cells at concentrations studied.Furthermore, these figures indicate that after compound addition(colcemid or paclitaxel), the cell indices for H460 cells firstdecreased with time and then increased, showing that H460 cells hadcomplex kinetic responses to either colcemid and paclitaxel. It isnoteworthy that cell index curves shown in FIG. 1A for H460 cells underthe influence of dibenzyl trisulfide (DBTS) at concentration of 25 μMand above are somewhat similar to the curves in FIGS. 1B and 1C, i.e.,after addition of DBTS (25 μM and above), the cell indices for H460cells also first decreased with time and then increased.

In another example, FIG. 6B shows the time-dependent cell index for A431(epidermoid cancer cell line) cells prior to and after addition of5-fluorouracil at various concentrations. As shown in FIG. 6B,5-fluorouracil exhibited inhibitory ability against the proliferation ofA431 cells at concentrations of 12.5 μM and above. The time dependentcell index curves in FIG. 6B are significantly different from those inFIG. 6A.

In another example, FIG. 13 shows the cell index data of HepaG-2 celllines under the influence of dibenzyl trisulfide. As shown in FIG. 13,dibenzyl trisulfide did not demonstrate anti-proliferation ability onHepaG-2 cells.

In Vivo Screening for Anticancer Activity

To evaluate the in vivo anticancer efficacy of the test compoundsincluding DBTS and ACEA100108 (a derivative of DBTS, see Table 33),various mouse models were used, including the mouse sarcoma S180 model,the mouse Lewis lung cancer model, P388 lymphocytic leukemia model, andthree human tumor xenograft models in immunodeficient nude mice: Bcap-37human breast cancer, HCT-8 human colon cancer, ao12/17 human ovariancancer. Details of the in vivo anticancer efficacy of the test compoundsare provided below.

Assessment of Acute Toxicity of DBTS and Compound ACEA100108

To evaluate the in vivo acute, intravenous toxicity of DBTS andACEA100108 (a derivative of DBTS, see Table 33), the experiments wereperformed in non-tumor bearing, normal Kunming mice by monitoring theacute response of mice to a single dose of DBTS or ACEA100108 viaintravenous injection (i.v.). The number of death for the treated micewas monitored and recorded. LD₅₀ values for these compounds werecalculated. Details of the study are provided below.

The following examples are offered to illustrate but not to limit theinvention.

Example 1 Anticancer Activity of DBTS Against Mouse Sarcoma S180 andMouse Lewis Lung Cancer

To evaluate the in vivo anticancer efficacy of the test compounds, twomouse transplanted tumor models were used for the in vivo evaluation:the mouse sarcoma S180 model and the mouse Lewis lung cancer model.Experimental mice were maintained in the Pharmacology Lab of ShanghaiPharmaceutical Industry Institute. The mouse source and specificationsare as follows. The mice were C57BL/6 and Kunming strains, provided byAcademic Sinica, Experimental Animal Center, and certification number:Academic Sinica Experimental Animal Certificate, No. 5. The mouse weightis between 18-20 g. Both male and female mice were used. However, foreach experiment, animals of same sex were used. The number of animalstested were as follows: 30 mice for the test compound group, including10 for the high dose group, 10 for the middle dose group and 10 for thelow dose group; 10 mice were for the positive compound group; 20 micefor the negative control group, including 10 mice for the Normal Salinegroup and 10 mice for the solvent only group. The high, middle and lowdoses of DBTS are, 50, 25 and 12.5 mg/kg/d, respectively.

Test controls. For the negative control, two groups were set up: thesolvent only control group and normal saline control group. In thesolvent only control group, each mouse was administered intravenouslywith the solvent only having the same volume and same concentration (10%for the sarcoma S180 model and 5% for the Lewis lung cancer model) asthose used for high dose DBTS test, once a day, and for 7 or 10consecutive days. In the normal saline group, each mouse wasadministered with 0.5 ml of normal saline, once per day and for 7 or 10consecutive days. For the positive control group, the anticancercompound, cyclophosphamide (CTX) was administered intraperitoneally at30 mg/kg, once per day and for 7 or 10 consecutive days.

Preparation and Administration of Test Compounds. Test compoundsolutions for evaluating anti-tumor efficiency cancer models wereprepared as follows. In the mouse sarcoma S180 mouse model, 200 mg ofDBTS was dissolved in 10 mL of castor oil (in polyoxyethlated version)first, and then mixed with 90 mL of normal saline. The final DBTSconcentration in the solution is 0.2%, and the final solventconcentration is 10%. Each mouse was administered intravenously with thecompound solution of 0.5 mL (high dose), 0.3 mL (middle dose) and 0.15mL (low dose), respectively.

In the mouse Lewis lung cancer model, 200 mg of DBTS was dissolved in 5mL castor oil (in polyoxyethlated version). Each time before use, thissolution was diluted with normal saline to achieve final DBTSconcentration of 0.2% (high dose), 0.1% (middle dose) and 0.05% (lowdose) respectively. In this case, each mouse (about 20 g in weight) wasadministered intravenously with 0.5 mL of the compound solution of agiven compound concentration. The intravenous injection speed was about0.5 mL/0.5 min.

The dosages and administration of test compounds are within theknowledge of those commonly skilled in pharmacology. For example, thetest compounds may be administered by intravenous injection with a testcompound solution twice per day and for 7 consecutive days.Alternatively, the test compounds may be administered by intravenousinjection with a test compound solution once per day and for 10consecutive days.

Preparation of Tumor Cells for Transplantation and Determination ofCompound Efficacy. To prepare the tumor cells, the fast grown tumorswere first removed from the transplanted tumor mice (the sarcoma S180model or the Lewis lung cancer model), the tumor tissues were dissected,and the tumor cell suspensions were prepared from the dissected tissuesat the concentration of 2-4×10⁷ tumor cells/ml. 0.2 mL of the tumor cellsuspension (between 4 and 8 million tumor cells) was then transplantedback into an experimental mouse by subcutaneous injection. Twenty fourhours after the transplantation, mice were administered intravenouslywith a given dose of DBTS, with normal saline, or solvent only whichserved as the negative control, or with 50 mg/kg CTX intraperitoneallywhich served as the positive control. Two weeks after thetransplantation, mice were sacrificed and the transplanted tumors wereremoved from the experimental mice. Each removed solid tumor wasweighted, and the tumor inhibition rate in the DBTS-treated groups andin the CTX-treated group was calculated according to the formula:

Tumor inhibition rate %=(average weight of tumor in the negative controlgroup−average weight of tumor in the compound treated group)/averageweight of tumor in the negative control group×100  (2)

For the mouse sarcoma S180 model, the S180 cells were subcutaneouslytransplanted at approximately 5 million cells per mouse. After 24 hoursof the transplantation, each mouse in the test group was administeredintravenously with dibenzyl trisulfide at 50, 25, or 12.5 mg/kgrespectively per day and for 7 or 10 consecutive days. For the positivecontrol group, each mouse was administered with cyclophosphamide(Cytoxan, CTX) at 50 mg/kg intraperitoneally per day and for 7consecutive days. For the negative control group, each mouse wasadministered intravenously either with normal saline, or with thesolvent for dibenzyl trisulfide at the same concentration as that in thetest group per day and for consecutive 7 days. For each group, 10 micewere used.

For the mouse Lewis lung cancer model, the Lewis lung cancer cells weresubcutaneously transplanted at approximately 5 million cells per mouse.After 24 hours of the transplantation, each mouse in the test group wasadministered intravenously with dibenzyl trisulfide at 50, 25, or 12.5mg/kg per day and for 10 consecutive days. For the positive controlgroup, each mouse was administered with CTX at 50 mg/kgintraperitoneally per day and for 10 consecutive days. For the negativecontrol group, each mouse was administered intravenously either withnormal saline, or with the solvent for dibenzyl trisulfide at the sameconcentration as that in the test group per day and for consecutive 10days. For each group, 10 mice were used.

Results. In the mouse sarcoma S180 model, DBTS showed an average tumorinhibition rate of 63.30%, 54.68% and 48.69% for the 50, 25 and 12.5mg/kg dosage groups respectively (relative to the normal salinecontrol). The detailed results are shown in Table 9 and FIG. 14,describing an in vivo efficacy study of 0.2% DBTS in the mouse sarcomaS180 model. In FIG. 14, the seven rows (1-7, respectively) representresults from the following administered compounds (iv×7qd): 1) negativecontrol; 2) normal saline; 3) DBTS (25 ml/kg); 4) DBTS (15 ml/kg); 5)DBTS (7.5 ml/kg); 6) solvent control (15 ml/kg) and 7) positive controlCTX (30 mg/kg).

It was observed that right after the intravenous injection of DBTS, miceexhibited transient abnormal reactions including jumping, fastbreathing, and lying down followed by reduced activities. Such reactionstypically lasted 10-15 minutes. The same abnormal reactions were alsoseen in the mice intravenously injected with only solvent. Therefore,the injection speed and the high concentration of the solvent other thanDBTS may result in the transient abnormal reactions in the mice.

In the Lewis lung cancer model, DBTS showed an average tumor inhibitionrate of 67.05%, 51.34% and 45.21% for the 50, 25 and 12.5 mg/kg dosagegroups respectively (relative to the normal saline control). Thedetailed results are summarized in Table 10 and FIG. 15, describing anefficacy study of 0.2% DBTS on mouse Lewis lung cancer. In FIG. 15, theseven rows (1-7, respectively) represent results from the followingadministered compounds: 1) negative control; 2) normal saline; 3) DBTS(25 ml/kg); 4) DBTS (15 ml/kg); 5) DBTS (7.5 ml/kg); 6) solvent control(15 ml/kg) and 7) positive control CTX (30 mg/kg). DBTS and the solventcontrol were administered iv×10 qd; the positive control wasadministered ip×7qd. In contrast to the mice used for the mouse sarcomaS180 experiment, the mice intravenously injected with either DBTS orsolvent in this experiment showed much minor transient abnormalreactions.

By using the solvent only as the negative control, the average in vivotumor inhibition rates of DTBS for the S180 sarcoma are 50.25%, 38.58%and 30.46% in 50, 25 and 12.5 mg/kg dosage groups respectively, as shownin Table 11. For the Lewis lung cancer model, the average in vivo tumorinhibition rates of DBTS are 62.28%, 44.30% and 37.38% in the 50, 25 and12.5 mg/kg dosage groups respectively, as shown in Table 12.

The results generated from two mouse transplanted tumor modelsdemonstrate the specific inhibition of transplanted tumor growth in themice administered intravenously with DBTS. When intravenouslyadministered with a high dose of DBTS (50 mg/kg/d, and for 7 or 10consecutive days), a tumor inhibition rate of 65% was achieved in eithermouse transplanted tumor model, by using the normal saline as thenegative control. The solvent used to prepare DBTS solution showed aweak inhibitory effect on the tumor growth in the mouse transplantedtumor models, and may also cause transient abnormal reactions in miceafter intravenous injection.

TABLE 9 In vivo antitumor efficacy of DBTS in the mouse sarcoma S180model (subcutaneously transplanted sarcoma) Animal weight DosageAdministration Animal number (g) Tumor weight Inhibition Sample(mg/kg/d) method (beginning/end) beginning/end (g) X +/− SD rate (%)DBTS 50 iv X 7 qd 10/10 19.5/22.9 0.98 ± 0.20

63.30 DBTS 25 iv X 7 qd 10/10 19.4/23.8 1.21 ± 0.14

54.68 DBTS 12.5 iv X 7 qd 10/10 19.4/24.5 1.37 ± 0.12

48.69 Positive 30 ip X 7 qd 10/10 19.6/20.3 0.22 ± 0.11

91.76 control (CTX) Negative Normal iv X 7 qd 10/10 19.3/24.8 2.67 ±0.15 control saline

p < 0.01, as compared with the negative control.

TABLE 10 The in vivo antitumor efficacy of DBTS in the mouse Lewiscancer model (subcutaneously transplanted tumor) Animal Tumor InhibitionDosage Administration Animal number weight (g) weight (g) rate Sample(mg/kg/d) method (beginning/end) beginning/end X +/− SD (%) DBTS 50 iv X10 qd 10/10 18.9/22.9  0.86 ± 0.14

67.05 DBTS 25 iv X 10 qd 10/10 19.3/23.5  1.27 ± 0.22

51.34 DBTS 12.5 iv X 10 qd 10/10 19.0/23.9  1.43 ± 0.18

45.21 Positive 30 ip X 10 qd 10/10 19.1/20.2 0.323 ± 0.14

87.62 control (CTX) Negative Normal iv X 10 qd 20/20 19.2/24.9  2.61 ±0.25 control saline

p < 0.01, as compared with the negative control.

TABLE 11 The in vivo antitumor efficacy of DBTS in the mouse sarcomaS180 model (subcutaneously transplanted tumor) Animal Tumor InhibitionDosage Administration Animal number weight (g) weight (g) rate Sample(mg/kg/d) method (beginning/end) beginning/end X +/− SD (%) DBTS 50 iv X7 qd 10/10 19.5/22.9 0.98 ± 0.20

50.25 DBTS 25 iv X 7 qd 10/10 19.4/23.8 1.21 ± 0.14

38.58 DBTS 12.5 iv X 7 qd 10/10 19.4/24.5 1.37 ± 0.12

30.46 Positive 30 ip X 7 qd 10/10 19.6/20.3 0.22 ± 0.11

88.83 control (CTX) Negative 10% solvent iv X 7 qd 10/10 19.3/24.7 1.97± 0.18 control

p < 0.01, as compared with the solvent (10%) only negative control.

TABLE 12 The in vivo antitumor efficacy of DBTS in the mouse Lewiscancer model (subcutaneously transplanted tumor) Animal weight TumorInhibition Dosage Administration Animal number (g) weight (g) rateSample (mg/kg/d) method (beginning/end) beginning/end X +/− SD (%) DBTS50 iv X 10 qd 10/10 18.9/22.9  0.86 ± 0.14

62.28 DBTS 25 iv X 10 qd 10/10 19.3/23.5  1.27 ± 0.22

44.30 DBTS 12.5 iv X 10 qd 10/10 19.0/23.9  1.43 ± 0.18

37.28 Positive 30 ip X 7 qd 10/10 19.1/20.2 0.323 ± 0.14

85.83 control (CTX) Negative 5% solvents iv X 7 qd 20/20 19.1/24.3  2.28± 0.25 control

p < 0.01, as compared with 5% solvent only negative control.

Example 2 Anticancer Activity of DBTS on Mouse Lewis Lung Cancer

This study evaluates the in vivo anticancer efficacy of dibenzyltrisulfide (DBTS) in the mouse Lewis lung cancer model as in Example 1.The experimental mice were maintained in the Pharmacology Lab ofShanghai Pharmaceutical Industry Institute. The mice for experimentswere C₅₇BL/6 strain, provided by Academic Sinica, Experimental AnimalCenter, certification number: SCXK (Shanghai) 2003-0003. The mouseweight was between 18 and 20 g. Only female mice were used. The numbersof animals tested were as follows: 10 for each dose group, 10 forpositive control group and 20 for negative control group (10 forphysiological control group and 10 for solvent-control group).

Test control. For the negative control, two groups were set up: thesolvent only control group and normal saline control group. In thesolvent only control group, each mouse was administered intravenouslywith the solvent only having the same volume and same concentration (5%solvent in normal saline) as those used in a high dose DBTS test, once aday, for 7-10 consecutive days. In the normal saline group, each mousewas administered with 0.5 ml of normal saline, once a day, for 10consecutive days. For positive control group, an anticancer compound,cyclophosphamide (Cytoxan, CTX, for intraperitoneal use) wasadministered intraperitoneally at 30 mg/kg, once a day for 7 consecutivedays. In addition, as a reference group, an anticancer compound, Taxol,was administered intravenously at 15, 10 and 7.5 mg/kg, once a day for 5consecutive days.

Preparation and Administration of Test Compounds. 400 mg of DBTS wasdissolved in 10 mL of castor oil (solvent) to have a DBTS concentration40 mg/ml in the solvent. Each time before use, this solution was dilutedin normal saline to achieve desired DBTS concentrations. Normal salinewas added to dilute DBTS solution to desired concentrations of 0.2%(high dose), 0.1% (middle dose) and 0.05% (low dose) respectively. Eachmouse was administered intravenously with the compound solution of 0.5mL at a controlled injection speed of 0.5 ml/0.5 min. 24 hrs after thetumor transplantation, intravenous injections of compound solutions intocarrier mice were performed once a day, for consecutive 7 or 10 days.

Preparation of Tumor Cells for Transplantation and Determination ofCompound Efficacy. To prepare the tumor cells, the fast growing tumorswere first removed from the transplanted tumor mice, the tumor tissueswere dissected and tumor cell suspensions were prepared in normal salineto have a concentration of 2-4×10⁷ cells/ml. 0.2 ml of cell suspensionwas subcutaneously injected into the axillary region of each mouse.Twenty four hours after the transplantation, mice were administered witha given doses of DBTS, with normal saline, or solvent only which servesas the negative control, or with 30 mg/kg CTX intraperitoneally whichserved as the positive control. About two weeks after transplantation,mice were sacrificed and the transplanted tumors were removed fromexperimental mice. Each removed solid tumor was weighed; the tumorinhibition rate in each dosage group was calculated according toequation (2) in Example 1 (Anticancer Activity of DBTS Against MouseSarcoma S180 and Mouse Lewis Lung Cancer).

For the mouse Lewis lung cancer model, the Lewis lung cancer cells weresubcutaneously transplanted at approximately 6 million cells per mouse.After 24 hours of the transplantation, each mouse in the test group wasadministered intravenously with dibenzyl trisulfide at 50, 25, or 12.5mg/kg per day and for 10 consecutive days. For the positive controlgroup, each mouse was administered with CTX at 30 mg/kgintraperitoneally per day and for 7 consecutive days. For the negativecontrol group, each mouse was administered intravenously either withnormal saline, or with the solvent for dibenzyl trisulfide at the sameconcentration as that in the test group per day and for consecutive 10or 7 days. For each group, 10 mice were used. For Taxol reference group,each mouse in the test group was administered intravenously with Taxolat 15, 10 or 7.5 mg/kg per day and for 5 consecutive days.

Results. In the Lewis lung cancer model, DBTS showed an average tumorinhibition rate of 65.77%, 51.61% and 43.10% for the 50, 25 and 12.5mg/kg dosage groups respectively (relative to the normal salinecontrol). The detailed results are shown in Table 13. By using thesolvent only as the negative control, the corresponding tumor inhibitionrates are 61.02%, 46.94% and 35.10%, respectively (Table 14). It wasobserved that right after the intravenous injection of DBTS, miceexhibited transient abnormal reactions including jumping, fastbreathing, and lying down followed by reduced activities. Such reactionstypically lasted 10-15 minutes. The same abnormal reactions were alsoseen in the mice intravenously injected with only solvent.

In the reference test, Taxol showed an average tumor inhibition rate of48.94%, 36.97 and 30.28% for the 15, 10 and 7.5 mg/kg dosage groupsrespectively (relative to the normal saline control). The detailedresults are shown in Table 15.

The result generated in the mouse Lewis lung cancer model demonstratesthe specific inhibition of transplanted tumor growth in the miceadministered intravenously with DBTS. When intravenously administeredwith a high dose of DBTS (50 mg/kg/d, and for 10 consecutive days), atumor inhibition rate of 65% was achieved in the mouse transplantedtumor model, by using the normal saline as the negative control. Suchdata have been shown to be reproducible. The solvent used to prepareDBTS solution showed a weak inhibitory effect on the tumor growth in themouse transplanted tumor models, and may also cause transient abnormalreactions in mice after intravenous injection.

TABLE 13 In vivo antitumor efficacy of DBTS in the mouse Lewis cancermodel (subcutaneously transplanted tumor). Animal Tumor DosageAdministration Animal No. weight (g) Tumor weight (g) Inhibition Sample(mg/kg/d) method beginning/end beginning/end X ± SD rate (%) DBTS 50ivx10 qd 10/10 21.0/23.4 0.955 ± 0.20*** 65.77 DBTS 25 ivx10 qd 10/1021.2/23.7  1.35 ± 0.10*** 51.61 DBTS 12.5 ivx10 qd 10/10 20.9/24.1  1.59± 0.16*** 43.01 Positive 30 ipx7 qd 10/10 21.1/22.3 0.258 ± 0.09***90.75 Control (CTX) Negative Normal ivx10 qd 20/20 21.3/26.0 2.79 ±0.30  Control saline ***P < 0.01, as compared with the negative control.

TABLE 14 In vivo antitumor efficacy of DBTS in the mouse Lewis cancermodel (subcutaneously transplanted tumor). Animal Tumor weight TumorDosage Administration Animal Number weight (g) (g) inhibition Sample(mg/kg/d) method Beginning/end beginning/end X ± SD rate (%) DBTS 50ivx10 qd 10/10 21.0/23.4 0.955 ± 0.20***  61.02 DBTS 25 ivx10 qd 10/1021.2/23.7 1.35 ± 0.10*** 46.94 DBTS 12.5 ivx10 qd 10/10 20.9/24.1 1.59 ±0.16*** 35.10 Negative 5% solvent ivx7 qd 10/10 21.3/26.0 2.79 ± 0.30  Control ***P < 0.01, as compared with the 5% solvent only negativecontrol

TABLE 15 In vivo antitumor efficacy of Taxol in the mouse Lewis cancermodel (subcutaneously transplanted tumor). Data is used as referencehere. Animal Animal weight Tumor weight Tumor Dosage Administrationnumber (g) (g) Inhibition Sample (mg/kg/d) method beginning/endbeginning/end X ± SD rate % Taxol 15 ivx5 qd 8/8 18.9/19.3 1.45 ±0.14*** 48.94 Taxol 10 ivx5 qd 8/8 18.7/21.7 1.79 ± 0.09*** 36.97 Taxol7.5 ivx5 qd 8/8 18.5/22.9 1.98 ± 0.14*** 30.28 Negative Normal ivx5 qd16/16 18.6/24.9 2.84 ± 0.31   Control saline ***P < 0.01, as comparedwith the negative control Note: Taxol is often used as positive controlfor anticancer efficacy test. The dosage is 10 mg/kg/d, ivx7 qd.

Example 3 In Vivo Anticancer Activity of ACEA100108 on Lewis Lung Cancerand P388 Lymphocytic Leukemia in Mice, and on Bcap-37 Human BreastCancer and HCT-8 Human Colon Cancer in Nude Mice

To evaluate the in vivo anticancer efficacy of compound ACEA100108 (aDBTS derivative, see Table 33), mouse models with transplanted cancerwere used, including Lewis lung cancer model and P388 lymphocyticleukemia model, and two human tumor xenograft models in immunodeficientnude mice: Bcap-37 human breast cancer and HCT-8 human colon cancer. Allthe mouse models are maintained in the Pharmacology Lab of ShanghaiPharmaceutical Industry Institute. For human tumor xenograft models,cancer cells were passed twice in vivo before being transplanted intothe nude mice for the study. Cultured human cancer cells in flask werefirst xenograft-transplanted in immunodeficient nude mice. After thecancer cells grew to a tumor of certain sizes in the nude mice, thetumor was removed form the nude mice and tumor tissues were dissected.The cell suspensions were prepared from the dissected tumor tissue andtransplanted back to immunodeficient nude mice again (i.e. the secondpassage of cancer cells in human cancer xenograft-transplanted model).After the cancer cells grew to certain size, the tumor was removed fromnude mice and the tumor tissues were dissected. The cell suspensionswere prepared from dissected tissues and were used for the study ofhuman cancer xenograft models described here.

The mice for experiments were C₅₇BL/6, DBF1 and BALB/c nude micestrains, provided by Academic Sinica, Experimental Animal Center,certification number: SCXK (Shanghai) 2003-0003. The mouse weight wasbetween 18 and 22 g. Both male and female mice were used. However, foreach experiment, animals of same sex were used. For the mousetransplanted tumor model, the numbers of animals tested were as follows:10 for each dose group, 10 for positive control group and 20 fornegative control group. For human tumor xenograft model, the numbers ofanimals tested were as follows: 6 for each dose group, 6 for positivecontrol group and 12 for negative control group.

Test control. For negative control, each mouse was administeredintravenously with the solvent only having the same volume and sameconcentration as those used in high dose ACEA100108 test, once a day,for 7 consecutive days. For positive control group, an anticancercompound, Taxol was administered intravenously at 10 mg/kg, once a dayfor 7 consecutive days. In a reference group, DBTS was administeredintravenously at 50 mg/kg, once a day for 7 consecutive days.

Preparation and Administration of Test Compounds. Compound ACEA100108was dissolved in hydrogenated castor oil (solvent) to have a compoundACEA100108 concentration of 20 mg/ml in the solvent. Each time beforeuse, this solution was diluted in normal saline to achieve desiredACEA100108 concentrations. Each mouse (about 20 g in weight) wasadministered intravenously with the compound solution of 0.5 mL at acontrolled injection speed of 0.5 ml/0.5 min. 24 hrs after the tumortransplantation, intravenous injections of compound solutions intocarrier mice were performed once a day, for consecutive 7 or 10 days.Different dosages of compound ACEA100108 between 100 and 6.25 mg/kg wereused in the study.

Preparation of Tumor Cells for Transplantation and Determination ofCompound Efficacy. To prepare the cancer cells for mouse Lewis lungcancer model, human breast cancer xenograft model and human colon cancerxenograft model, the fast growing tumors were first removed from thetransplanted tumor mice. The tumor tissues were dissected and tumor cellsuspensions were prepared in normal saline to have a concentration of2-4×10⁷ cells/ml. 0.2 ml of cell suspension was subcutaneously injectedinto the axillary region (right-side) of each mouse. Twenty four hoursafter the transplantation, mice were administered with a given dose ofACEA100108, or with solvent only which serves as the negative control,or with 10 mg/kg Taxol which served as positive control, or with 50mg/kg DBTS which served as a reference test. Between two and four weeksafter transplantation, mice were sacrificed and the transplanted tumorswere removed from experimental mice. Each removed solid tumor wasweighed; the tumor inhibition rate in each dosage group was calculatedaccording to equation (2) in Example 1.

For human tumor xenograft model, all used materials, including animalfood, animal cage, supporting materials and apparatus contacted byanimals, were high-pressure sterilized. Nude mice were maintained inlaminar flow shelves under SPF condition. After tumor transplantation,mouse weight and tumor size in each compound dosage group weredynamically monitored and plotted. The tumor size was determined bymeasuring the major axis (a) and minor axis (b) of the tumor, and tumorvolume was calculated according to the formula

Tumor volume=a×b ²/2  (3)

To prepare cancer cells for the P388 murine lymphocytic leukemia model,ascites of a P388 leukemia-bearing mouse were removed under sterilecondition. The ascites were diluted in normal saline (1:6 for ascites tonormal saline) to prepare cell suspension. 0.2 mL of the cell suspensionwas then injected intraperitoneally. Twenty four hours aftertransplanting the cancer cells into mice, mice were administered withgiven doses of a given dose of ACEA100108, or with solvent only whichserves as the negative control, or with 10 mg/kg Taxol and with 2 mg/kgMMC (mitomycin C) which served as positive controls, or with 50 mg/kgDBTS which served as a reference test. The life span of carrier mice wasdetermined within 30 days. The life span ratio comparing to the negativecontrol group of the carrier mice in each compound treatment group wascalculated according to the formula:

Life span ratio %=average life span for the compound treatmentgroup/average life span for the negative control group×100%  (4)

Results. In the Lewis lung cancer model, ACEA100108 showed the averageof in vivo tumor inhibition rates of 60.15%, 55.35% and 34.32%,respectively, in 100 (administered only 5 times because of toxicity), 25and 6.25 mg/kg dosage groups (relative to the solvent-only control). Inthe same experiment, DBTS showed the average in vivo tumor inhibitionrates of 63.10% and 57.93%, respectively, in 100 (administered only 5times because of toxicity) and 25 mg/kg dosage groups, and Taxol showedan in vivo tumor inhibition rate of 43.91% for the routineadministration dosage of 10 mg/kg. The results are summarized in Table16.

In murine lymphocytic leukemia model, the average increase in life spanof mice treated with compound ACEA100108 were 106.18%, 107.22% and109.28%, respectively, in 50, 25 and 12.5 mg/kg dosage groups. In thesame experiment, the average increase in life span of mice was 109.28%for the mice being treated with DBTS compound at a dosage of 50 mg/kg,and the average increase in life span of mice treated with 10 mg/kgTaxol compound was 109.28%. The details are provided in Table 17.

In Bcap-37 human breast cancer xenograft model in nude mice, ACEA100108showed the average in vivo tumor inhibition rates of 64.13%, 56.10% and31.40%, respectively, in 50, 25 and 8 mg/kg dosage groups. In the sameexperiment, DBTS showed the average in vivo tumor inhibition rate of66.98% for a 50 mg/kg dosage and Taxol showed an average in vivo tumorinhibition rate of 48.84% for the routine administration dosage of 10mg/kg. The details are provided in Table 18 and FIG. 16, describing anefficacy study of DBTS and ACEA 100108 on Bcap-37 human breast cancerxenograft-transplanted in nude mice. In FIG. 16, the seven rows (1-7,respectively) represent results from the following administeredcompounds: 1) negative control; 2) solvent; 3) ACEA 100108 (50 mg/kg);4) ACEA 100108 (20 mg/kg); 5) ACEA 100108 (8 mg/kg); 6) DBTS (50 mg/kg);and 7) positive control (taxol, 10 mg/kg). The test compounds andcontrols were administered iv×7qd. The dynamic changes of tumor size aresummarized in Table 19 and FIG. 17. The dynamic change of body weight ofcarrier mice results are summarized in Table 20 and FIG. 18.

In HCT-8 human lung cancer xenograft model in nude mice, ACEA100108showed the average in vivo tumor inhibition rates of 45.62%, 28.10% and15.03%, respectively, in 50, 25 and 8 mg/kg dosage groups. In the sameexperiment, DBTS showed the average in vivo tumor inhibition rate of46.08% for a 50 mg/kg dosage and Taxol showed an average in vivo tumorinhibition rate of 33.33% for the routine administration dosage of 10mg/kg. The details are provided in Table 21 and FIG. 19, describing anefficacy study of DBTS and ACEA 100108 on HCT-8 human colon cancerxenograft transplanted in nude mice. In FIG. 19, the seven rows (1-7,respectively) represent results from the following administeredcompounds: 1) negative control; 2) solvent; 3) ACEA 100108 (50 mg/kg);4) ACEA 100108 (20 mg/kg); 5) ACEA 100108 (8 mg/kg); 6) DBTS (50 mg/kg);and 7) positive control (taxol, 10 mg/kg). The test compounds andcontrols were administered iv×7qd. The dynamic changes of tumor size aresummarized in Table 22 and FIG. 20. The dynamic change of body weight ofcarrier mice results are summarized in Table 23 and FIG. 21.

Based on the results from the in vivo evaluation of two mouse tumormodels and two humor tumor xenograft models, ACEA100108 may beeffectively administered at 50 mg/kg and iv×7qd. In addition, theanticancer effect of ACEA100108 on mouse Lewis lung cancer model andBcap-37 human breast cancer model is stronger than its effect on HCT-8human colon cancer model. However, ACEA100108 did not exhibit anticancereffect on P388 mouse leukemia model. Furthermore, for the same dosageand same drug-administration procedure, the anticancer effect for abovemodels of compound ACEA100108 is comparable with that of DBTS, and isbetter than that of Taxol under routine treatment dosage conditions.

TABLE 16 The in vivo antitumor efficacy of compound ACEA100108 in themouse Lewis cancer model by subcutaneous seeding. Animal weight DosageAdministration Animal No. (g) Tumor weight (g) Inhibition Sample mg/kg/dmethod beginning/end beginning/end X ± SD rate (%) ACEA100108 100 ivx7qd 10/8  20.6/23.4 1.08 ± 0.17*** 60.15 ACEA100108 25 ivx7 qd 10/1020.1/24.2 1.21 ± 0.22*** 55.35 ACEA100108 6.25 ivx7 qd 10/10 20.3/24.41.78 ± 0.24*** 34.32 ACEA100101 100 ivx7 qd 10/6  20.7/23.1 1.00 ±0.15*** 63.10 ACEA100101 25 ivx7 qd 10/10 20.5/23.6 1.14 ± 0.17*** 57.93Positive control 10 ivx7 qd 10/10 20.4/23.8 1.52 ± 0.15*** 43.91 (Taxol)Negative control Solvent ivx7 qd 20/20 20.3/24.7 2.71 ± 0.26   ***P <0.01, as compared with the negative control group

TABLE 17 The in vivo antitumor efficacy of compound ACEA100108 in themurine P388 lympholytic leukemia model (transplanted by injection ofcancer cells into the peritoneal cavity of host mice). Beginning dosageadministration animal No. animal weight Average life span Life spanSample mg/kg/d method beginning/end (g) X ± SD ratio % ACEA100108 50ivx7 qd 10/0 20.4 10.3 ± 0.95 106.18 ACEA100108 25 ivx7 qd 10/0 20.410.4 ± 1.17 107.22 ACEA100108 12.5 ivx7 qd 10/0 20.1 10.6 ± 0.84 109.28ACEA100101 50 ivx7 qd 10/0 20.7 10.6 ± 1.26 109.28 Positive control 10ivx7 qd 10/0 20.2 10.5 ± 1.18 108.25 (Taxol) Positive control 2 ivx7 qd10/1 20.6 18.1 ± 0.15*** 186.59 (MMC) Negative control Solvent ivx7 qd20/0 20.0  9.7 ± 0.66 ***p < 0.01, as compared with negative controlgroup. Note: In general, a compound is regarded as having antitumorefficacy when the life span ratio of carrier mice in the treatment groupis more than 125%.

TABLE 18 The in vivo antitumor efficacy of compound ACEA100108 onBcap-37 human breast cancer that was xenograft-transplanted inimmunodeficient nude mice by subcutaneous implanting. animal TV No.animal weight tumor weight tumor Tumor inhibition dosage Administrationbeginning/ (g) (g) inhibition Volume rate % Sample mg/kg/d method endbeginning/end X ± SD rate % TV (cm³) T/C ACEA100108 50 ivx7 qd 6/618.1/23.0 0.617 ± 0.09*** 64.13 0.310 17.67 ACEA100108 20 ivx7 qd 6/617.6/23.0 0.755 ± 0.09*** 56.10 0.488 27.82 ACEA100108 8 ivx7 qd 6/618.0/23.0  1.18 ± 0.23*** 31.40 0.985 56.15 ACEA100101 50 ivx7 qd 6/618.0/22.8 0.568 ± 0.07*** 66.98 0.196 11.17 Positive 10 ivx7 qd 6/618.1/23.3  0.88 ± 0.17*** 48.84 0.685 39.05 control Taxol NegativeSolvent ivx7 qd 12/12 18.0/23.5 1.72 ± 0.19  1.754 Control ***P < 0.01,as compared with negative control group.

TABLE 19 The dynamic change in tumor size in the in vivo antitumorefficacy test of compound ACEA100108 on Bcap-37 human breast cancer thatwas xenograft-transplanted in immunodeficient nude mice by subcutaneousimplanting. Tumor volume (cm³) Dosage Administration Days after tumortransplantation Sample mg/kg method 7 days 14 d 21 d 24 d NegativeSolvent ivx7 qd 0.01 10/12†† 0.254 ± 0.06 0.858 ± 0.06 1.754 ± 0.37Control Taxol 10 mg/kg ivx7 qd 0.01 4/6†† 0.065 ± 0.02 0.249 ± 0.070.685 ± 0.14 ACEA100108 50 mg/kg ivx7 qd 0.01 3/6†† 0.023 ± 0.02 0.112 ±0.03  0.31 ± 0.05 ACEA100108 20 mg/kg ivx7 qd 0.01 5/6†† 0.049 ± 0.030.167 ± 0.03 0.488 ± 0.07 ACEA100108  8 mg/kg ivx7 qd 0.01 4/6†† 0.068 ±0.02 0.214 ± 0.04 0.985 ± 0.4  ACEA100101 50 mg/kg ivx7 qd 0.01 2/6††0.014 ± 0.01 0.079 ± 0.01 0.196 ± 0.02 10/12††: it means that out oftotal 12 mice, 10 had tumor size sufficiently large when one touchesthese mice, one can feel tumor in each mouse.

TABLE 20 The dynamic change in body weight of carrier mice in the invivo antitumor efficacy test of compound ACEA100108 on Bcap-37 humanbreast cancer that was xenograft- transplanted in immunodeficient nudemice by subcutaneous implanting. dosage administration Body weight ofmice (g) Samples (mg/kg/d) method 0 day 7 day 14 d 21 d 24 d NegativeSolvent ivx7 qd   18 ± 0.9 20.2 ± 0.9 21.9 ± 1.1 23.2 ± 1.2 23.5 ± 0.9Control Taxol 10 ivx7 qd 18.1 ± 1.1 19.7 ± 1 21.7 ± 1 22.8 ± 1.2 23.3 ±1.2 ACEA100108 50 ivx7 qd 18.1 ± 1.1 18.8 ± 0.8 20.5 ± 1 22.2 ± 1.2   23± 1.2 ACEA100108 20 ivx7 qd 17.6 ± 0.8 19.7 ± 1 20.8 ± 1 22.7 ± 1.2   23± 1.2 ACEA100108  8 ivx7 qd   18 ± 0.6 19.5 ± 1 21.5 ± 1 22.3 ± 1.6   23± 1.5 ACEA100101 50 ivx7 qd 18 ± 1 18.7 ± 0.8 20.3 ± 0.8   22 ± 0.6 22.8± 1.6

TABLE 21 The in vivo antitumor efficacy of compound ACEA100108 on HCT-8human colon cancer that was xenograft-transplanted in immunodeficientnude mice by subcutaneous implanting. Animal weight tumor Tumor TVdosage administration Animal No. (g) tumor weight (g) inhibition volumeinhibition Sample (mg/kg/d) method beginning/end beginning/end X ± SDrate % TV (cm³) rate % T/C ACEA100108 50 iv × 7 qd 6/6 18.3/20.3 0.832 ±0.10*** 45.62 0.525 33.63 ACEA100108 20 iv × 7 qd 6/6 18.5/22.5 1.10 ±0.23  28.10 0.654 41.89 ACEA100108  8 iv × 7 qd 6/6 18.8/22.5 1.30 ±0.23  15.03 0.870 55.73 ACEA100101 50 iv × 7 qd 6/6 18.2/23.0 0.825 ±0.07*** 46.08 0.502 32.17 Positive 10 iv × 7 qd 6/6  18.7/22.58  1.02 ±0.11*** 33.33 0.694 44.45 control Taxol Negative Solvent iv × 7 qd 12/1218.8/23.9 1.53 ± 0.23  1.561 control ***P < 0.01, as compared withnegative control.

TABLE 22 The dynamic change in tumor size in the in vivo antitumorefficacy test of compound ACEA100108 on HCT-8 human colon cancer thatwas xenograft-transplanted in immunodeficient nude mice by subcutaneousimplanting. Tumor volume (cm³) Dosage Administration Days aftertransplantation Sample (mg/kg/d) method 7 day 14 d 21 d 25 d Negativesolvent iv × 7 qd 0.01 12/12†† 0.253 ± 0.07 0.911 ± 0.2  1.561 ± 0.26control Taxol 10 iv × 7 qd 0.01 6/6†† 0.116 ± 0.03 0.308 ± 0.06 0.694 ±0.15 ACEA100108 50 iv × 7 qd 0.01 6/6†† 0.112 ± 0.02 0.236 ± 0.02 0.525± 0.14 ACEA100108 20 iv × 7 qd 0.01 6/6†† 0.122 ± 0.04 0.317 ± 0.050.654 ± 0.09 ACEA100108  8 iv × 7 qd 0.01 6/6†† 0.166 ± 0.05 0.379 ±0.04  0.87 ± 0.15 ACEA100101 50 iv × 7 qd 0.01 4/6†† 0.031 ± 0.02 0.204± 0.03 0.502 ± 0.18 4/6††: it means that out of total 6 mice, 4 hadtumor size sufficiently large when one touched these mice, one couldfeel the tumor.

TABLE 23 The dynamic change in body weight of carrier mice in the invivo antitumor efficacy test of compound ACEA100108 on HCT-8 human coloncancer that was xenograft-transplanted in immunodeficient nude mice bysubcutaneous implanting. Dosage administration Mouse Body weight (g)Sample (mg/kg/d) method 0 Day 7 d 14 d 21 d 25 d Negative Solvent iv × 7qd 18.8 ± 1 20.5 ± 0.7 21.8 ± 1.1 22.4 ± 0.9 22.9 ± 1.1 control Taxol 10iv × 7 qd 18.7 ± 1 19.8 ± 1.2 21.7 ± 1.2 22.2 ± 0.8 22.5 ± 1.8ACEA100108 50 iv × 7 qd 18.3 ± 1 17.7 ± 1.2 17.8 ± 2.3 19.5 ± 1.6 20.3 ±1 ACEA100108 20 iv × 7 qd 18.5 ± 1 19.5 ± 1 20.7 ± 1 21.8 ± 1.2 22.5 ± 1ACEA100108  8 iv × 7 qd 18.8 ± 0.8 19.7 ± 0.8 21.3 ± 1.2 21.8 ± 1.2 22.5± 1 ACEA100101 50 iv × 7 qd 18.2 ± 1.2 18.8 ± 1 19.5 ± 1 20.7 ± 0.8   23± 1.3

Example 4 In Vivo Anticancer Activity of ACEA100108 on ao10/17 HumanOvarian Cancer in Nude Mice

To evaluate the in vivo anticancer efficacy of compound ACEA100108, anao10/17 human ovarian cancer xenograft model in immunodeficient nudemice was used. The cell line and mice were maintained in thePharmacology Lab of Shanghai Pharmaceutical Industry Institute. For theao10/17 human ovarian cancer xenograft models, cancer cells were passedtwice in vivo before being transplanted into the nude mice for thestudy. In another word, cultured human ovarian cancer ao10/17 cells inflask were first xenograft-transplanted in immunodeficient nude mice.After the cancer cells grew to a tumor of certain sizes in the nudemice, the tumor was removed form the nude mice and tumor tissues weredissected. The cell suspensions were prepared from the dissected tumortissue and transplanted back to immunodeficient nude mice again (i.e.the second passage of cancer cells in human cancerxenograft-transplanted model). After the cancer cells grew to certainsize, the tumor was removed from nude mice and the tumor tissues weredissected. The cell suspensions were prepared from dissected tissues andwere used for the study of human cancer xenograft models described here.

The mice for experiments were C₅₇BL/6, DBF1 and BALB/c (nude mice)strains, provided by Academic Sinica, Experimental Animal Center,certification number: SCXK (Shanghai) 2003-0003. The mouse weight wasbetween 18 and 22 g. Only female mice were used in this study. For humantumor xenograft model, the numbers of animals tested were as follows: 6for each dose group, 6 for positive control group and 12 for negativecontrol (solvent only) group. The high, middle and low doses ofACEA100108 were 50, 25 and 8 mg/kg/d, respectively.

Test control. For negative control, each mouse was administeredintravenously with the solvent only having the same volume and sameconcentration as those used in high dose ACEA100108 test, once a day,for 7 consecutive days. For positive control group, an anticancercompound, Taxol was administered intravenously at 10 mg/kg, once a dayfor 7 consecutive days. In a reference group, DBTS was administeredintravenously at 50 mg/kg, once a day for 7 consecutive days.

Preparation and Administration of Test Compounds. Compound ACEA100108was dissolved in hydrogenated castor oil (solvent) to have a compoundACEA100108 concentration of 20 mg/ml in the solvent. Each time beforeuse, this solution was diluted in normal saline to achieve desiredACEA100108 concentrations. Each mouse (about 20 g in weight) wasadministered intravenously with the compound solution of 0.5 mL at acontrolled injection speed of 0.5 ml/0.5 min. 24 hrs after the tumortransplantation, intravenous injections of compound solutions intocarrier mice were performed once a day, for consecutive 7 days. Thehigh, middle and low dose of compound ACEA100108 was 50, 20 and 8 mg/kg,respectively.

Preparation of Tumor Cells for Transplantation and Determination ofCompound Efficacy. To prepare the cancer cells for human ovarian cancerxenograft model, the fast growing tumors were first removed from thetransplanted tumor mice. The tumor tissues were grounded in normalsaline (1:6 for tumor volume to saline volume) and tumor cellsuspensions were prepared in the normal saline. 0.2 ml of cellsuspension was subcutaneously injected into the axillary region(right-side) of each mouse. Twenty four hours after the transplantation,mice were administered with a given dose of ACEA100108, or with solventonly which serves as the negative control, or with 10 mg/kg Taxol whichserved as positive control, or with 50 mg/kg DBTS which served as areference test. Between two and four weeks after transplantation, micewere sacrificed and the transplanted tumors were removed fromexperimental mice. Each removed solid tumor was weighed; the tumorinhibition rate in each dosage group was calculated according toequation (2) in Example 1.

For the human ovarian cancer xenograft model, all used materials,including animal food, animal cage, supporting materials and apparatuscontacted by animals, were high-pressure sterilized. Nude mice weremaintained in laminar flow shelves under SPF condition. After tumortransplantation, mouse weight and tumor size in each compound dosagegroup were dynamically monitored and plotted. The tumor size wasdetermined by measuring the major axis (a) and minor axis (b) of thetumor, and tumor volume was calculated according to the equation (3) inExample 3.

Results. In ao10/17 human ovarian cancer xenograft model in nude mice,ACEA100108 showed the average in vivo tumor inhibition rates of 53.40%,46.67% and 33.19%, respectively, in 50, 25 and 8 mg/kg dosage groups. Inthe same experiment, DBTS showed the average in vivo tumor inhibitionrate of 57.30% for a 50 mg/kg dosage and Taxol showed an average in vivotumor inhibition rate of 45.39% for the routine administration dosage of10 mg/kg. The details are provided in Table 24 and FIG. 22, describingan efficacy study of DBTS and ACEA 100108 on ao10/17 human ovariancancer xenograft-transplanted in nude mice. In FIG. 22, the seven rows(1-7, respectively) represent results from the following administeredcompounds: 1) negative control; 2) solvent; 3) ACEA 100108 (50 mg/kg);4) ACEA 100108 (20 mg/kg); 5) ACEA 100108 (8 mg/kg); 6) DBTS (50 mg/kg);and 7) positive control (taxol, 10 mg/kg). The test compounds andcontrols were administered iv at 7qd.

The dynamic changes of tumor size are summarized in Table 25 and FIG.23. The dynamic change of body weight of carrier mice results aresummarized in Table 26 and FIG. 24. For the same dosage and samedrug-administration procedure, the anticancer effect of compoundACEA100108 in ao10/17 human ovarian cancer models is comparable withthat of compound ACEA100101, and is better than that of Taxol underregular treatment dosage conditions.

TABLE 24 The in vivo antitumor efficacy of compound ACEA100108 onao10/17 human ovarian cancer xenograft transplanted in immunodeficientnude mice (subcutaneously transplanted tumor). Body weight Tumor WeightTumor Dosage Administration Animal No. (g) (g) Inhibition Sample mg/kg/dMethod Beginning/end Beginning/end X ± SD Rate % ACEA100108 50 iv × 7 qd6/6 17.2/21.3 0.657 ± 0.13*** 53.40 ACEA100108 20 iv × 7 qd 6/617.2/22.0 0.752 ± 0.12*** 46.67 ACEA100108  8 iv × 7 qd 6/6 17.7/22.20.942 ± 0.14*** 33.19 ACEA100101 50 iv × 7 qd 6/6 17.3/21.3 0.602 ±0.10*** 57.30 Positive Control 10 iv × 7 qd 6/6 17.8/22.5  0.77 ±0.12*** 45.39 (Taxol) Negative Control Solvent iv × 7 qd 12/12 17.8/23.01.41 ± 0.17  ***P < 0.01, as compared with negative control.

TABLE 25 The dynamic change in tumor size in the in vivo antitumorefficacy test of compound ACEA100108 on ao10/17 human ovarian cancerthat was xenograft-transplanted in immunodeficient nude mice(subcutaneously transplanted tumor). Tumor volume (cm³) DosageAdministration Days after tumor transplantation Sample mg/kg method 7 D14 d 21 d 24 d Negative Solvent iv × 7 qd 0.01 ± 7/12†† 0.213 ± 0.030.985 ± 0.03 1.648 ± 0.22 Control Taxol 10 iv × 7 qd 0.01 ± 2/6†† 0.033± 0.02 0.196 ± 0.03 0.349 ± 0.08 ACEA100108 50 iv × 7 qd 0.01 ± 1/6†† 0.01 ± 6/6†† 0.148 ± 0.02 0.316 ± 0.06 ACEA100108 20 iv × 7 qd 0.01 ±2/6††  0.03 ± 0.02 0.206 ± 0.03 0.402 ± 0.1  ACEA100108  8 iv × 7 qd0.01 ± 3/6†† 0.048 ± 0.03 0.249 ± 0.05  0.89 ± 0.39 ACEA100101 50 iv × 7qd 0.01 ± 2/6††  0.01 ± 6/6†† 0.129 ± 0.01 0.225 ± 0.04 7/12††: it meansthat out of total 12 mice, 7 had tumor size sufficiently large when onetouched the mouse, one could feel the tumor.

TABLE 26 The dynamic change in body weight of carrier mice in the invivo antitumor efficacy test of compound ACEA100108 on ao10/17 humanovarian cancer that was xenograft- transplanted in immunodeficient nudemice by subcutaneous implanting. Dosage Administration Body weight ofmice (g) Sample (mg/kg) method 0 d 7 d 14 d 21 d 24 d Negative Solventiv × 7 qd 17.8 ± 1.1 20.3 ± 0.8 20.9 ± 0.9 21.9 ± 0.9 23.0 ± 1 ControlTaxol 10 iv × 7 qd 17.8 ± 1.2   19 ± 0.9 20.2 ± 1.2 21.0 ± 0.9 22.5 ± 1ACEA100108 50 iv × 7 qd 17.2 ± 0.8 16.7 ± 0.8 17.8 ± 1.5 19.3 ± 1 21.3 ±1.2 ACEA100108 20 iv × 7 qd 17.2 ± 1.2 18.3 ± 0.8 19.8 ± 0.8 20.5 ± 122.0 ± 1.4 ACEA100108  8 iv × 7 qd 17.7 ± 0.5   19 ± 0.6 20.5 ± 1 21.2 ±0.8 22.2 ± 0.8 ACEA100101 50 iv × 7 qd 17.3 ± 1 17.3 ± 1.4 18.8 ± 1.219.5 ± 1 21.3 ± 1.2

Example 5 In Vivo Anticancer Activity of ACEA100108 on Bcap-37 HumanBreast Cancer in Nude Mice

To evaluate the in vivo anticancer efficacy of compound ACEA100108,Bcap-37 human breast cancer xenograft model in immunodeficient nude micewas used. The cell line and mouse model are maintained in thePharmacology Lab of Shanghai Pharmaceutical Industry Institute. For theBcap-37 human breast cancer xenograft models, cancer cells were passedtwice in vivo before being transplanted into the nude mice for thestudy. In another word, cultured human breast cancer Bcap-37 cells inflask were first xenograft-transplanted in immunodeficient nude mice.After the breast cancer cells grew to a tumor of certain sizes in thenude mice, the tumor was removed form the nude mice and tumor tissueswere dissected. The cell suspensions were prepared from the dissectedtumor tissue and transplanted back to immunodeficient nude mice again(i.e. the second passage of cancer cells in human cancerxenograft-transplanted model). After the cancer cells grew to certainsize, the tumor was removed from nude mice and the tumor tissues weredissected. The cell suspensions were prepared from dissected tissues andwere used for the study of human cancer xenograft models described here.

The mice for experiments were BALB/c (nude mice) strains, provided byAcademic Sinica, Experimental Animal Center, certification number: SCXK(Shanghai) 2003-0003. The mouse weight was between 18 and 22 g. Onlyfemale mice were used in this study. For human tumor xenograft model,the numbers of animals tested were as follows: 6 for each dose group, 6for positive control group and 12 for negative control (solvent only)group. The high, middle and low doses of ACEA100108 were 50, 25 and 8mg/kg/d, respectively.

Test control. For negative control, each mouse was administeredintravenously with the solvent only having the same volume and sameconcentration as those used in high dose ACEA100108 test, once a day,for 7 consecutive days. For positive control group, an anticancercompound, Taxol was administered intravenously at 10 mg/kg, once a dayfor 7 consecutive days.

Preparation and Administration of Test Compounds. Compound ACEA100108was dissolved in hydrogenated castor oil (solvent) to have a ACEA100108concentration of 20 mg/ml in the solvent. Each time before use, thissolution was diluted in normal saline to achieve desired ACEA100108concentrations. Each mouse (about 20 g in weight) was administeredintravenously with the compound solution of 0.5 mL at a controlledinjection speed of 0.5 ml/0.5 min. Seven days after the tumortransplantation, the transplanted tumors grew to size sufficiently largethat could be felt by hands when one touched the animal. From that timeon, intravenous injections of compound solutions into carrier mice wereperformed once a day, for consecutive 7 or 10 days. The high, middle andlow dose of ACEA100108 was 50, 20 and 8 mg/kg, respectively.

Preparation of Tumor Cells for Transplantation and Determination ofCompound Efficacy. To prepare the cancer cells for human breast cancerxenograft model, the fast growing tumors were first removed from thetransplanted tumor mice. The tumor tissues were grounded in normalsaline (1:6 for tumor volume to saline volume) and tumor cellsuspensions were prepared in the normal saline having cell concentrationof 2-4×10⁷ cells/ml. 0.2 ml of cell suspension was subcutaneouslyinjected into the axillary region (right-side) of each mouse. Aboutseven days after the transplantation, tumors in the mice grewsufficiently large so that tumor could be felt by hands when one touchedthe animals. From that time on, mice were administered with a given doseof ACEA100108, or with solvent only which serves as the negativecontrol, or with 10 mg/kg Taxol which served as positive control.Between three and four weeks after transplantation, mice were sacrificedand the transplanted tumors were removed from experimental mice. Eachremoved solid tumor was weighed; the tumor inhibition rate in eachdosage group was calculated according to equation (2) in Example 1.Based on the tumor volume, another parameter, namely, tumor volumeinhibition rate was also calculated, according to

T/C(%)=average volume of tumor in the compound treated group/averageweight of tumor in the negative control group×100%  (5)

For the human breast cancer xenograft model, all used materials,including animal food, animal cage, supporting materials and apparatuscontacted by animals, were high-pressure sterilized. Nude mice weremaintained in laminar flow shelves under SPF condition. After tumortransplantation, mouse weight and tumor size in each compound dosagegroup were dynamically monitored and recorded. The tumors size wasdetermined by measuring the major axis (a) and minor axis (b) of thetumor, and tumor volume was calculated according to the equation (3) inExample 3.

Results. In Bcap-37 human breast cancer xenograft model in nude mice,ACEA100108 showed the average in vivo tumor inhibition rates of 52.24%,47.31% and 28.21%, respectively, in 50, 20 and 8 mg/kg dosage groupswhen the compound was administered according to iv×7qd procedure.Furthermore, it showed the average in vivo tumor inhibition rates of56.92% for 50 mg/kg dosage when the compound was administered accordingto 10×qd procedure. In the same experiment, Taxol showed an average invivo tumor inhibition rate of 44.33% for the routine administrationdosage of 10 mg/kg. The details are provided in Table 27 and FIG. 25,describing an efficacy study of ACEA 100108 on Bcap-37 human breastcancer xenograft-transplanted in nude mice. In FIG. 25, the seven rows(1-7, respectively) represent results from the following administeredcompounds: 1) negative control; 2) solvent; 3) ACEA 100108 (50 mg/kg);4) ACEA 100108 (20 mg/kg); 5) ACEA 100108 (8 mg/kg); 6) ACEA 100108 (50mg/kg); and 7) positive control (taxol, 10 mg/kg). The test compoundsand controls were administered iv at 7qd, except for ACEA 1001008 at 50mg/kg, which was administered iv×10qd. The result of tumor volumeinhibition rates are shown in Table 28. The dynamic changes of tumorsize are summarized in Table 26. The dynamic change of body weight ofcarrier mice results are summarized in Table 27.

In the Bcap-37 human breast cancer xenograft model in nude mice,ACEA100108 showed a tumor inhibition rate above 50% for a compoundadministration procedure in which compound was administered after thetumor grew to sufficient large so that the tumor could be felt by hands.Furthermore, when dosing times of the compound in the nude miceincreased, there was no apparent increased toxic effect to mice, whilethere was increased tumor inhibition. In addition, the middle dosage ofACEA100108 administered here into nude mice showed a better anticancerefficacy than that of the routine treatment dosage of Taxol.

TABLE 27 The in vivo antitumor efficacy of compound ACEA100108 onBcap-37 human breast cancer xenograft transplanted in immunodeficientnude mice (subcutaneously transplanted tumor). (Based on tumor weight).Tumor Body weight Tumor Weight Inhibition Dosage Administration AnimalNo. (g) (g) Rate Sample mg/kg/d Method Beginning/end Beginning/end X ±SD C − T/C % ACEA100108 50 iv × 7 qd 6/6 18.2/22.8 0.745 ± 0.10*** 52.24ACEA100108 20 iv × 7 qd 6/6 18.8/24.3 0.822 ± 0.12*** 47.31 ACEA100108 8 iv × 7 qd 6/6 18.5/24.0  1.12 ± 0.18*** 28.21 ACEA100108 50 iv × 10qd 6/6 18.8/21.2 0.672 ± 0.10*** 56.92 Positive Control 10 iv × 7 qd 6/618.8/24.3  0.92 ± 0.07*** 41.03 (Taxol) Negative Control Solvent iv × 7qd 12/12 18.6/24.8  1.56 ± 0.14 ***P < 0.01, as compared with negativecontrol.

TABLE 28 The in vivo antitumor efficacy of compound ACEA100108 onBcap-37 human breast cancer xenograft transplanted in immunodeficientnude mice (subcutaneously transplanted tumor). (Based on tumor volume)Tumor Body weight Tumor Volume Volume Dosage Administration Animal No.(g) (g) Inhibition Sample mg/kg/d Method Beginning/end Beginning/end X ±SD T/C % ACEA100108 50 iv × 7 qd 6/6 18.2/22.8 0.485 ± 0.06*** 26.91ACEA100108 20 iv × 7 qd 6/6 18.8/24.3 0.740 ± 0.18*** 41.06 ACEA100108 8 iv × 7 qd 6/6 18.5/24.0 0.962 ± 0.23*** 53.38 ACEA100108 50 iv × 10qd 6/6 18.8/21.2 0.280 ± 0.04*** 15.53 Positive Control 10 iv × 7 qd 6/618.8/24.3 0.799 ± 0.23*** 44.33 (Taxol) Negative Control Solvent iv × 7qd 12/12 18.6/24.8 1.802 ± 0.43 ***P < 0.01, as compared with negativecontrol.

TABLE 29 The dynamic change in tumor size in the in vivo antitumorefficacy test of compound ACEA100108 on Bcap-37 human breast cancer thatwas xenograft-transplanted in immunodeficient nude mice (subcutaneouslytransplanted tumor). Tumor volume (cm³) Dosage Administration Days aftertumor transplantation Sample mg/kg method 7 d 14 d 21 d 24 d NegativeSolvent iv × 7 qd 0.01 12/12†† 0.282 ± 0.07 0.962 ± 0.25 1.802 ± 0.43Control Taxol 10 iv × 7 qd 0.01 6/6†† 0.103 ± 0.02 0.283 ± 0.05 0.799 ±0.23 ACEA100108 50 iv × 7 qd 0.01 6/6†† 0.049 ± 0.02 0.169 ± 0.03 0.485± 0.06 ACEA100108 20 iv × 7 qd 0.01 6/6†† 0.087 ± 0.01  0.23 ± 0.04 0.74 ± 0.18 ACEA100108  8 iv × 7 qd 0.01 6/6†† 0.107 ± 0.02  0.27 ±0.03 0.962 ± 0.23 ACEA100108 50 iv × 10 qd 0.01 6/6†† 0.048 ± 0.03 0.114± 0.02  0.28 ± 0.04 12/12††: it means that out of total 12 mice, all hadtumor size sufficiently large when one touched the mouse, one could feelthe tumor.

TABLE 30 The dynamic change in body weight of carrier mice in the invivo antitumor efficacy test of compound ACEA100108 on Bcap-37 humanbreast cancer that was xenograft- transplanted in immunodeficient nudemice by subcutaneous implanting. Dosage Administration Body weight ofmice (g) Sample (mg/kg) method 0 d 7 d 14 d 21 d 24 d Negative Solventiv × 7 qd 18.6 ± 0.9 21.1 ± 1.2 21.9 ± 1.2 23.5 ± 1.2 24.8 ± 1.1 ControlTaxol 10 iv × 7 qd 18.8 ± 1 20.5 ± 1 21.5 ± 1.6 23.0 ± 1.4 24.3 ± 0.8ACEA100108 50 iv × 7 qd 18.2 ± 1.2 19.3 ± 1 20.7 ± 1.2 21.8 ± 1.5 22.8 ±1.5 ACEA100108 20 iv × 7 qd 18.8 ± 1.2 19.5 ± 1 21.2 ± 1.5 22.3 ± 1.624.3 ± 0.8 ACEA100108  8 iv × 7 qd 18.5 ± 1 19.8 ± 1.5 21.7 ± 1.4 23.2 ±1.5   24 ± 1.4 ACEA100108 50 iv × 10 qd 18.8 ± 1 19.7 ± 1 20.3 ± 1.421.5 ± 1.8 21.2 ± 1.2

Example 6 Acute Toxicity Test of DBTS and Compound ACEA100108Determination of the Intravenous Injection LD₅₀ in Mice

The experiments to test DBTS and ACEA100108 acute toxicity wereperformed in mice. The test mice were randomly divided into six groups(five dosing groups and one control group). Each group contained 20Kunming strain mice, and among them, 50% were male and 50% were female.After administration of a single dose of DBTS or ACEA100108 viaintravenous injection (i.v.), the acute response to DBTS or ACEA100108compound, and the death of the treated mice during the first two weekswere monitored and recorded. The LD₅₀ value was calculated using theBliss method. The mouse single i.v. dose LD₅₀ value of DBTS was 258.53mg/kg (234.96 to 284.46 mg/kg), and the mouse single i.v. dose LD₅₀value of ACEA100108 was 316 mg/kg (284.26-351.28 mg/kg).

Materials and Method. The test chemical compound were DBTS andACEA100108, which were dissolved into hydrogenated castor oil in thepre-warmed water bath and made as a 20 mg/ml solution. The solution wasfurther diluted to desired experiment concentrations with the normalsaline. The administration volume was 0.5 ml i.v. per mouse and theinjection speed was 0.5 ml/0.5 min.

The experimental mice were Kunming strain, provided by the ExperimentalAnimal Department, Shanghai Pharmaceutical Industry Institute. Thecertificate number of the facility was Animal Facility CertificationNumber 107. The average weight of the mice was 18-20 gram. Each testgroup contained 20 Kunming strain mice, and among them, 10 mice weremale and 10 mice were female. Five experimental doses were used, whichwere 400 mg/kg, 320 mg/kg, 256 mg/kg, 204.8 mg/kg and 163.8 mg/kg. Themice in the control group were only given the same volume of thesolvent, which were diluted hydrogenated castor oil. All the testingmice were given a single intravenous injection of DBTS, ACEA100108, orthe solvent that served as the control at the injection speed of 0.5ml/0.5 min. The acute response to DBTS, ACEA100108 or the solventimmediately after the administration, as well as weight change, and thedeath within the first two weeks of the administration were monitoredand recorded. The intravenous injection LD₅₀ values in mice werecalculated using the Bliss method.

Result. Immediately after intravenous injection, mice showed behavioralabnormalities, which included jumping, running, convulsion, andshortness of breath (accelerated respiration). At high dose groups, somemice died of convulsive seizure within 3 min after the injection. Thedeath occurred within one hour of the administration and the peak was atthe 12^(th) hour of the administration. No pathological abnormality inthe organs of the dead mice was found by autopsy. The survival miceshowed no severe toxic symptoms except early reduced activities andloose hair, which were gradually recovered, and there was no delayedtoxic manifestations seen within the 14 day following up monitoring.Although survival mice were healthy and behaved normal, the mice showedweight loss to some degree. Based experimental data, the mouse singlei.v. dose LD₅₀ value of DBTS was 258.53 mg/kg (234.96 to 284.56 mg/kg),and the mouse single i.v. dose LD₅₀ value of ACEA100108 was 316 mg/kg(284.26-351.28 mg/kg). There was no significant difference in LD₅₀values between male mice and female mice (p value>0.05). The acutetoxicity results for DBTS and ACEA100108 were summarized in Tables 31and 32. To evaluate the possible toxic effect of the solvent on themice, the mice in the control group were administered with the samevolume of the solvent. The mice given the solvent showed early abnormalmanifestations and weight loss to a degree less than the mice dosed withDBTS or ACEA100108. This suggests that the acute toxic effects seen inthe dosing mice are related to DBTS or ACEA100108.

TABLE 31 Acute toxicity in the Kunming mice given a single intravenousinjection dose of DBTS. Distribution of dead animals on each day afterthe single Percentage LD₅₀ Average animal Dosage intravenous injectionof dead (95% CL) weight (g) Sex Mg/kg Animal number 1 2 3 4 5 6 7 8 9 10. . . 14 animals % g/kg Beginning End Male 400 10 10 0 0 0 0 0 0 0 0 0 .. . 0 100 261.08 20.1 — 320 10 7 1 1 0 0 0 0 0 0 0 . . . 0 90(230.3~295.9) 20.1 25.0 256 10 2 1 1 0 0 0 0 0 0 0 . . . 0 40 20.4 26.3204.8 10 0 0 1 0 0 0 0 0 0 0 . . . 0 10 19.9 26.6 163.8 10 0 0 0 0 0 0 00 0 0 . . . 0 0 20.0 26.9 Female 400 10 10 0 0 0 0 0 0 0 0 0 . . . 0 100256   19.8 — 320 10 5 2 1 0 0 0 0 0 0 0 . . . 0 80 (221.7~295.5) 20.624.5 256 10 3 1 0 1 0 0 0 0 0 0 . . . 0 50 19.9 24.2 204.8 10 0 1 0 1 00 0 0 0 0 . . . 0 20 20.5 24.3 163.8 10 0 0 0 0 0 0 0 0 0 0 . . . 0 019.9 24.5 50% 400 20 20 0 0 0 0 0 0 0 0 0 . . . 0 100 258.53 Male, 32020 12 3 2 0 0 0 0 0 0 0 . . . 0 85 (234.9~284.4) 50% 256 20 5 2 1 1 0 00 0 0 0 . . . 0 45 Female 204.8 20 0 1 1 1 0 0 0 0 0 0 . . . 0 15 163.820 0 0 0 0 0 0 0 0 0 0 . . . 0 0

TABLE 32 Acute toxicity in the Kunming mice given a single intravenousinjection dose of ACEA100108. Distribution of dead animals on each dayafter the single intravenous Percentage LD₅₀ Average animal DosageAnimal injection of dead (95% CL) weight Sex Mg/kg number 1 2 3 4 5 6 78 9 10 . . . 14 animals % g/kg Beginning End Male 500 10 10 0 0 0 0 0 00 0 0 . . . 0 100 319.3 20.3 — 400 10 3 3 0 1 0 0 0 0 0 0 . . . 0 70(271.9~375.0) 19.9 26.0 320 10 0 3 1 0 0 0 0 0 0 0 . . . 0 40 20.0 26.7256 10 0 2 0 1 0 0 0 0 0 0 . . . 0 30 20.2 26.3 204.8 10 0 0 1 0 0 0 0 00 0 . . . 0 10 20.3 26.3 163.8 10 0 0 0 0 0 0 0 0 0 0 . . . 0 0 20.427.0 Female 500 10 10 0 0 0 0 0 0 0 0 0 . . . 0 100 313.2 19.6 — 400 105 2 0 1 0 0 0 0 0 0 . . . 0 80 (272.7~359.7) 19.9 23.5 320 10 2 2 1 0 00 0 0 0 0 . . . 0 50 19.9 24.6 256 10 0 2 0 1 0 0 0 0 0 0 . . . 0 3020.1 24.6 204.8 10 0 0 0 0 0 0 0 0 0 0 . . . 0 0 20.5 24.3 163.8 10 0 00 0 0 0 0 0 0 0 . . . 0 0 19.6 24.2 50% 500 20 20 0 0 0 0 0 0 0 0 0 . .. 0 100 316   male, 400 20 8 5 0 2 0 0 0 0 0 . . . 0 75 (284.2~351.2)50% 320 20 2 5 2 0 0 0 0 0 0 . . . 0 45 female 256 20 0 4 0 2 0 0 0 0 00 . . . 0 30 204.8 20 0 0 1 0 0 0 0 0 0 0 . . . 0 5 163.8 20 0 0 0 0 0 00 0 0 0 . . . 0 0

Example 7 Inhibition of Cell Proliferation by DBTS, Colcemid andPaclitaxel

H460 cells (human lung cancer cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 8000 cells per well and were pre-incubated inincubator under standard cell culture conditions for about 22 hrs.Dibenzyl trisulfide (DBTS), colcemil and paclitaxel at differentconcentrations in DMSO were added into wells following the incubationperiod. The cell status was monitored prior to and after the compoundaddition using RT-CES system. The cell indexes of different wells werebetween 1.7 and 1.9 for DBTS and colcemid solutions just before thecompound addition, and between 1.4 and 1.9 for paclitaxel. FIGS. 1A-Cshow the normalized cell index as a function of time prior to and afterthe compound addition. The cell index was normalized against the cellindex values at a time point just after compound addition (about 23 hrsafter cell seeding).

Example 8 Inhibition of Cell Proliferation by DBTS in MV522 Cells

MV522 cells (human lung cancer cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 10,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 22 hrs.Dibenzyl trisulfide solutions in DMSO were added into wells followingthe incubation period. The cell status was monitored prior to and afterthe compound addition using RT-CES system. The cell indexes of differentwells were between 1.0 and 1.6 just before the compound addition. FIG. 2shows the normalized cell index as a function of time prior to and afterthe compound addition. The cell index was normalized against the cellindex values at a time point just after compound addition (about 23 hrsafter cell seeding).

Example 9 Inhibition of Cell Proliferation by Dibenzyl Trisulfide inMCF-7 Cells

MCF-7 cells (human breast cancer cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 10,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 44 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 1.2 and 1.5 just before the compound addition. FIG. 3shows the normalized cell index as a function of time prior to and afterthe compound addition. The cell index was normalized against the cellindex values at a time point just after compound addition (about 44.5hrs after cell seeding).

Example 10 Inhibition of Cell Proliferation by Dibenzyl Trisulfide inA549 Cells

A549 cells (human lung cancer cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 8,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 17 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 0.72 and 1.26 just before the compound addition. FIG.4 shows the normalized cell index as a function of time prior to andafter the compound addition. The cell index was normalized against thecell index values at a time point just after compound addition (about 18hrs after cell seeding).

Example 11 Inhibition of Cell Proliferation by Dibenzyl Trisulfide inPC3 Cells

PC3 cells (human prostate cancer cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 10,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 22.5 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 0.34 and 0.54 just before the compound addition. FIG.5 shows the normalized cell index as a function of time prior to andafter the compound addition. The cell index was normalized against thecell index values at a time point just after compound addition (about23.5 hrs after cell seeding).

Example 12 Inhibition of Cell Proliferation by DBTS and 5-Fluorouracilin A431 Cells

A431 cells (human epidermoid cancer cells) were seeded into wells ofmicrotiter plate devices (electronic plates, i.e., the plates comprisemicroelectrode sensor arrays in the wells of the plate) with an initialseeding density of 10,000 cells per well and were pre-incubated inincubator under standard cell culture conditions for about 22.3 hrs.Various concentrations of DBTS and 5-fluorouracil solutions were addedinto wells following the incubation period. The cell status wasmonitored prior to and after the compound addition using RT-CES system.The cell indexes of different wells of DBTS were between 0.6 and 1.2 forDBTS, and between 0.6 and 1.2 for 5-fluorouracil just before thecompound addition. FIGS. 6A-B show the normalized cell index as afunction of time prior to and after the compound addition. The cellindex was normalized against the cell index values at a time point justafter compound addition (22.6 hrs after cell seeding).

Example 13 Inhibition of Cell Proliferation by DBTS in HT1080 Cells

HT1080 cells (human fibrosarcoma cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 4,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 18.6 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 0.72 and 1.45 just before the compound addition. FIG.7 shows the normalized cell index as a function of time prior to andafter the compound addition. The cell index was normalized against thecell index values at a time point just after compound addition (about 20hrs after cell seeding).

Example 14 Inhibition of Cell Proliferation by DBTS in MDA-231 Cells

MDA-231 cells (human breast cancer cells) were seeded into wells of 16×or 96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 5,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 18.7 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 0.65 and 0.82 just before the compound addition. FIG.8 shows the normalized cell index as a function of time prior to andafter the compound addition. The cell index was normalized against thecell index values at a time point just after compound addition (about19.6 hrs after cell seeding).

Example 15 Inhibition of Cell Proliferation by DBTS in HT-29 Cells

HT-29 cells (human colon cancer cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 10,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 25 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 0.95 and 1.13 just before the compound addition. FIG.9 shows the normalized cell index as a function of time prior to andafter the compound addition (about 26 hrs after cell seeding). The cellindex was normalized against the cell index values at a time point justprior to compound addition.

Example 16 Inhibition of Cell Proliferation by DBTS in HC-2998 Cells

HC-2998 cells (human colon cancer cells) were seeded into wells of 16×or 96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 10,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 24.7 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 0.33 and 0.68 just before the compound addition. FIG.10 shows the normalized cell index as a function of time prior to andafter the compound addition. The cell index was normalized against thecell index values at a time point just after compound addition (about25.7 hrs after cell seeding).

Example 17 Inhibition of Cell Proliferation by DBTS in OVCAR4 Cells

OVCAR4 cells (human ovarian cancer cells) were seeded into wells of 16×or 96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 10,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 27 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 1.4 and 1.7 just before the compound addition. FIG.11 shows the normalized cell index as a function of time prior to andafter the compound addition. The cell index was normalized against thecell index values at a time point just after compound addition (about 28hrs after cell seeding).

Example 18 Inhibition of Cell Proliferation by DBTS in A2780 Cells

A2780 cells (human colon cancer cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 20,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 16.4 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell indexes of differentwells were between 2.2 and 3.7 just before the compound addition. FIG.12 shows the normalized cell index as a function of time prior to andafter the compound addition. The cell index was normalized against thecell index values at a time point just after compound addition (about17.5 hrs after cell seeding).

Example 19 Response of HepG2 Cells to DBTS

HepG2 cells (human hepatosarcoma cells) were seeded into wells of 16× or96× microtiter plate devices (electronic plates, i.e., the platescomprise microelectrode sensor arrays in the wells of the plate) with aninitial seeding density of 15,000 cells per well and were pre-incubatedin incubator under standard cell culture conditions for about 22 hrs.Dibenzyl trisulfide solution in DMSO was added into wells following theincubation period. The cell status was monitored prior to and after thecompound addition using RT-CES system. The cell index was between 0.7and 0.97 just before the compound addition. FIG. 13 shows the normalizedcell index as a function of time prior to and after the compoundaddition. The cell index was normalized against the cell index values ata time point just after compound addition (about 22.7 hrs after cellseeding). From the cell index data shown here, it appears that dibenzyltrisulfide exhibits no inhibition effect on HepG2 cell proliferation andno cytotoxic effect on the HepG2 cells within the exposing dose range.

Example 20 Inhibition of Cancer Cell Proliferation by DBTS and ItsDerivatives

The anticancer potency of DBTS and its derivatives were tested in 8different types of human cancer cell lines using the RT-CES system andMTT assay. The 8 cancer cell lines were HT1080 (the human fibrosarcomacell line), H460 (human non small cell lung cancer cell line), OVCAR4(the human ovarian cancer cell line), MCF7 (human breast cancer cellline) MDA-MB231 (M231, the human breast cancer cell line) A2780 (thehuman colon cancer cell line) Jurkat (the human T cell leukemia cellline). The test DBTS derivatives include ACEA100107, ACEA100108,ACEA100109, ACEA100111, ACEA100115, ACEA100116, ACEA100117, ACEA100118,ACEA100119, and ACEA100120. ACEA100129 was also tested in HT1080, HELAand MCF7 cells, having an IC₅₀ value of 0.82 μM, 0.42 μM and 2.3 μM,respectively. The chemical structures of the derivatives are shown inTables 33 and 34.

For the assay performed on the RT-CES system, the cells were seeded intothe 16× or 96× microtiter plate devices (electronic plates, i.e., theplates comprise microelectrode sensor arrays in the wells of the plate)at the seeding density ranging from 5000 cells/well to 15,000cells/well. The cells were incubated at 5% CO₂ and 37° C. for overnighttill the cell indices reached the growth phase where the cell index wasbetween 0.8 and 1.2. Serially diluted compounds were then added to thecells followed by dynamic monitoring of the effect of the compounds onthe cell proliferation and cytotoxicity. The time-dependent IC₅₀ valuesfor each derivative were calculated based the dose responses of cellindex value at different time points after compound treatment. The IC₅₀values shown in Table 35 corresponds to the time points at whichcompound showed the maximum inhibition after the treatment.

For the MTT assay, the cells were seeded into the regular 96× wellplates at the cell seeding density ranging from 5000 cells/well to15,000 cells/well. The cells were incubated at 5% CO₂ and 37° C. forovernight. The derivatives were serially diluted and added to the cells.The treatment was terminated after 48 hours of incubation by adding MTTstaining reagent. After 4 hours, the staining was stopped by the stopbuffer and then the calorimetric measurement was carried out on amicrotiter plate reader at dual wave length, 650 nm and 550 nm. The IC₅₀values for tested derivatives were calculated using the calorimetricreadouts and listed in Table 36.

TABLE 33

DBTS Derivative R DBTS (ACEA100101) H ACEA100108 p-F ACEA100118 p-ClACEA100115 o-Cl ACEA100116 m-Me ACEA100117 m-CF₃ ACEA100129 p-Me

TABLE 34

DBTS Derivative Name Ar ACEA100111

ACEA100107

ACEA100109

ACEA100120

ACEA100119

TABLE 35 IC₅₀ values (uM) of DBTS and its derivatives in 7 cancer celllines using the RT-CES system. Cell line ACEA100107 ACEA100108ACEA100109 ACEA100111 ACEA100115 HT1080 5.3 1.3 9.4 3.6 2.2 OVCAR4 5.1 210.5 12.2 0.5 M231 4.8 3.05 19.1 17.5 1.06 A2780 2.65 0.5 6.25 1.3 0.75H460 20 9.2 42.2 33.5 23.2 MCF7 5.6 2.15 8.8 6.25 7.8HepG2 >50 >50 >50 >50 >50 Cell line ACEA100116 ACEA100117 ACEA100118AECA100119 ACEA100120 DBTS HT1080 1.9 27.5 1.2 36 9 2.2 OVCAR4 0.6 33.52.25 34.8 11.5 1.75 M231 1.06 41 2.3 >50 16 2.4 A2780 0.75 11.8 0.7 17.44.4 0.4 H460 12.5 >50 9.6 31.5 18.2 11.1 MCF7 2.75 48.8 4.4 31.6 11.96.6 HepG2 >50 >50 >50 >50 >50 >50

TABLE 36 IC₅₀ values (uM) of DBTS and its derivatives in 8 cell linesusing the MTT assay. ACEA100107 ACEA100108 ACEA100109 ACEA100111ACEA100115 Jurkat 1.2 0.35 4.7 2.6 0.51 M231 6.6 4.4 >50 1.6 3.1 HT10805.3 19 20.8 34 12.4 A2780 1.05 4.7 6.3 5.35 1.5MCF-7 >50 >50 >50 >50 >50 OVCAR4 >50 >50 >50 >50 >50 H460 27 10.25 27.412.5 11.75 HepG2 >50 >50 >50 >50 >50 ACEA100116 ACEA100117 ACEA100118ACEA100119 ACEA100120 ACEA100101 Jurkat 0.3 8 0.5 >25 7.3 0.35 M231 0.6514.9 1.2 >50 9.7 2.4 HT1080 2.2 47.5 1.15 39.3 9 2.55 A2780 0.8 11.70.8 >50 15.2 1.2 MCF-7 >50 >50 >50 >50 >50 >50OVCAR4 >50 >50 >50 >50 >50 >50 H460 5.4 >50 7.95 >50 20.8 14.2HepG2 >50 >50 >50 >50 >50 >50

Example 21 Kinetic Inhibition of Cancer Cell Proliferation by ACEA100108

The anticancer potency of a DBTS derivative, ACEA100108 was tested in 7cancer cell lines on the RT-CEA system. The cell lines were HT1080,H460, OVCAR4, MCF7, MDA-MB231, HepG2, and A2780. The cancer cells wereseeded into 16× or 96× microtiter plate devices (i.e. electronic plates)containing wells at cell seeding density ranging from 5000 cells/well to15000 cells/well, and the seeded cells were then incubated at 37° C., 5%CO₂. The cancer cell growth was monitored in real time on the RT-CESsystem till the cells reached the growth phase, which takesapproximately 20 hours. Cells were then treated with ACEA100108 whichwere serially diluted at the concentrations ranging from 50 uM to 0.38uM. The inhibition of the cancer proliferation of ACEA100108 andcytotoxicity responses of various cell lines to ACEA100108 weremonitored on the RT-CES system in real time. The kinetic curves of thecell-compound interaction was then recorded and shown in the FIG. 26.The cell index curves were normalized against the cell index values at atime point just after compound addition (approximately 18-24 hrs aftercell seeding).

Example 22 Kinetic Inhibition of Cancer Cell Proliferation by the DBTSDerivatives

The kinetic inhibition of proliferation of HT1080 cancer cells andcytotoxicity effects of the DBTS derivatives on HT1080 cancer cells weremeasured on the RT-CES system. The DBTS derivatives are ACEA100107,ACEA100109, ACEA100111, ACEA100114, ACEA100115, ACEA100116, ACEA100117,ACEA100118, ACEA100119, and ACEA100120. The HT1080 cells (humanfibrosarcoma) were seeded into the wells of 16× or 96× microtiter platedevices (electronic plates) at the seeding density of 5000 cells/well.After 20 hour incubation at 5% CO₂ and 37° C. till the cells reached thegrowth phase, the serially diluted DBTS-derivatives at the concentrationranging from 50 uM to 0.38 uM were added to the cells, and the cellresponse to the DBTS derivatives was monitored and recorded in real timefor 48 hours on the RT-CES system. FIG. 27 shows the kinetic curves ofinteractions between cells and DBTS-derivatives at differentconcentrations. The cell index curves were normalized against the cellindex values at a time point just after compound addition (approximately18-24 hrs after cell seeding).

Example 23 Suppression of Microtubule Dynamics by DBTS and itsDerivative Compounds ACEA100108 and ACEA100116 Overview

Microtubules are important in numerous cellular processes, includingmitosis when the duplicated chromosomes are separated into two identicalsets before cleavage of the cell into two daughter cells. The key roleof microtubules and their dynamics in mitosis and cell division makemicrotubules an important target for anticancer drugs. In cells duringinterphase, microtubules exchange their tubulin with soluble tubulin inthe cytoplasmic pool with half times of ˜3 minutes to several hours.With the onset of mitosis, the interphase microtubule networkdisassembles and is replaced by a population of highly dynamicmicrotubules which forms the mitotic spindle and moves the chromosomes.Mitotic spindle microtubules are 20-50 times more dynamic thanmicrotubules in interphase cells, and some spindle microtubules exchangetheir tubulin with tubulin in the soluble pool with half-times as rapidas 15 seconds.

The dynamics of mitotic spindle microtubules are exquisitely sensitiveto modulation by regulators and to disruption by microtubule-activedrugs. Microtubule-targeted drugs can alter microtubule polymerizationand dynamics in a wide variety of ways. The mechanisms of action ofthree ACEA compounds designated as DBTS, ACEA100108, and ACEA100116 withrespect to (1) the ability to influence the microtubule network incultured cells, (2) the ability to influence microtubule assembly invitro and (3) the ability to influence microtubule dynamics in vitro,are described below.

Methods

Cell Culture and Immunocytochemistry. COS cells were grown in DMEM mediasupplemented with non-essential amino acids, 10% FBS,antibiotic-antimycotic (Gibco BRL) at 37° C. and 5.5% CO₂. Forimmunofluorescence microscopy, cells were plated on polylysine coatedcoverslips and treated with various concentrations of the three ACEAcompounds, paclitaxel or vinblastine for either 4 or 24 hours (seeindividual figures for concentrations used in any given experiment).Cells were then rinsed once with warm PBS, fixed with cold methanol,rinsed again in PBS and blocked overnight at 4° C. in PBT (PBS, 1% BSA,0.5% Triton X-100). All subsequent stains and washes were done in PBT atroom temperature unless stated otherwise. Cells were stained with theanti-tubulin mouse antibody DM-1 at 1:1000 for 1 hour, washed four timesfor 15 minutes each and then treated with Cy-3 conjugated goat antimouse antibody at 1:100 for 1 hour in the dark. Next, samples werewashed four times for 15 minutes each in PBT in the dark followed by afinal 15 minute wash in PBS in the dark. Samples were then viewed bylaser scanning confocal microscopy.

Microtubule Assembly Assays. Microtubule seeds were synthesized byincubating purified bovine brain tubulin with 1 mM GTP, 10% glycerol and10% DMSO at 35° C. for 30 minutes, followed by shearing by passing theassembled microtubules 6 times through a 27 gauge needle. Microtubuleassembly was assayed by adding 27.5 ul of microtubule seeds tospectrophotometer cuvettes (maintained at 35° C.) containing 247.5 ulpurified bovine brain tubulin in a PEM-100 buffer (100 mM Pipes pH=6.8,1 mM EDTA, 1 mM MgSO₄) supplemented with 1 mM GTP (and drug whereapplicable) and monitoring the OD₄₀₀ for 2 hours. Since the compoundsare dissolved in DMSO and DMSO can have a significant effect onmicrotubule assembly, DMSO was added to all cuvettes so as to equal thelargest volume of drug added to reactions. It should be noted that theinitial velocity of the microtubule assembly reactions is so fast thatone can not always catch the initial rise on the light scatteringprofile because it is occurring while samples are being prepared.However, all samples start at the same optical density, since they areidentical with the exception of the drug being introduced.

Tubulin Purification and Microtubule Dynamics Assays. Tubulin waspurified, as described in the literature (“Kinetic stabilization ofmicrotubule dynamic instability in vitro by vinblastine”, Toso, R. J.,Jordan, M. A., Farrell, K. W., Matsumoto, B. and Wilson, L.,Biochemistry, 1993, 32, 1285-1293). Briefly, microtubule-associatedprotein-rich bovine brain microtubule protein was prepared by threecycles of assembly and disassembly. Tubulin was purified from othermicrotubule proteins by elution through a Whatman P-11 phosphocellulosecolumn equilibrated in PEM50 (50 mM Pipes, 1 mM MgSO₄, 1 mM EGTA, 0.1 mMGTP). Purified tubulin (>99% pure) was drop-frozen in liquid nitrogenand stored at −70° C. Purified tubulin (15 μM tubulin dimer) waspolymerized at the ends of sea urchin (Strongylocentrotus purpuratus)axonemal seeds at 37° C. in the presence or absence of ACEA 01, 08 or 16in PMEM buffer (87 mM Pipes, 36 mM MES, 1.4 mM MgCl₂, 1 mM EDTA, pH 6.8)and 2 mM GTP. The dynamics of individual microtubules were recorded at37° C. using differential interference contrast enhanced videomicroscopy. The ends were designated as plus or minus on the basis ofthe growth rate, the number of microtubules that grew at opposite endsof the seeds, and the relative lengths of the microtubules (Panda, D.,Goode, B. L., Feinstein, S. C. and Wilson, L., Kinetic stabilization ofmicrotubule dynamics at steady state by tau and microtubule-bindingdomains of tau, Biochemistry, 1995, 34, 11117-11127; Walker, R. A.,O'Brien, E. T., Pryer, N. K., Soboeiro, M. F., Voter, W. A., Erickson,H. P. and Salmon, E. D., Dynamic instability of individual microtubulesanalyzed by video light microscopy: rate constants and transitionfrequencies, J. Cell Biol. 1988, 107, 1437-1448). Plus ends wereanalyzed for 10 minutes per slide during the steady-state phase ofpolymerization (˜45 min after initiation of polymerization). Lifehistories of individual microtubules were collected as described byPanda et al. 1995 (Panda, D., Goode, B. L., Feinstein, S. C. and Wilson,L., Kinetic stabilization of microtubule dynamics at steady state by tauand microtubule-binding domains of tau, Biochemistry, 1995, 3411117-11127) with modifications. Data points were collected at 1-3 sintervals.

A microtubule was considered to be growing or shortening if it increasedor decreased in length at a rate >0.5 μm/min. microtubules exhibitinggrowth rates of <0.5 μm/min over a period greater than 30 s wereconsidered to be in an attenuated state. Average rates, lengths anddurations are the averages of independent events. The catastrophefrequency was calculated by dividing the number of shortening events bythe total time of growth and attenuation tracked, and rescue frequencywas calculated by dividing the number of rescue events by the total timeof shortening tracked. To control for experimental error, each conditionwas filmed over multiple days using at least two distinct tubulin/GTPmixtures (2-3 slides each). No gross variation in microtubule dynamicswas observed between mixtures or slides of a given condition. Theconcentration of drug used in dynamic instability assays was chosen byinitially observing microtubules stabilized with half the concentrationused in microtubule assembly assays. If most microtubules on a slidewere stable, the concentration of drug would be reduced until any givenmicrotubule tracked would have at least two growth or shortening eventsin the span of 10 minutes.

FIGS. 28-38 show the effect of DBTS and organosulfur compounds ACEA100108 and ACEA100116 on microtubule network in cultured cells. FIG. 28shows images of microtubules in control cells exposed to no drugs. Themicrotubule networks appear as expected. FIG. 29 shows images ofmicrotubules in cells exposed to taxol for 4 hours. Microtubules appearbundled in some locations; with increasing concentration, bundlingappears more extensive but the microtubules often appear shorter than inthe control cells. FIG. 30 shows images of microtubules in cells exposedto taxol for 24 hours. With increasing dosage, microtubule abnormalitiesincrease. As this figure shows, there is increased bundling and theshort microtubules persist. Additionally, major cellular abnormalitiesbecome apparent.

FIG. 31 shows images of microtubules in cells exposed to vinblastine for4 hours. With increasing dosage, the microtubule network begins to fallapart and the microtubules become much shorter. FIG. 32 shows images ofmicrotubules in cells exposed to vinblastine for 24 hours. As thisfigure shows, major cell abnormalities are widespread in the microtubulenetwork.

FIG. 33 shows images of microtubules in cells exposed to DBTS for 4hours. The microtubule network is completely disrupted; only very shortmicrotubules exist and the overall level of tubulin in microtubulesappears to be significantly reduced. This effect could be quantitatedbiochemically by non-ionic detergent extraction and immunoblotting. FIG.34 shows images of microtubules in cells exposed to DBTS for 24 hours.At the lowest dosage, there are some microtubules present and the cellsappear to have partially recovered when compared to cells exposed to thedrug for only 4 hours; no cells are viable after treatment for 24 hourswith either 6 uM or 18 uM of DBTS.

FIG. 35 shows images of microtubules in cells exposed to ACEA100108 for4 hours. Similar to DBTS, the microtubule network is markedly altered atall concentrations tested. Microtubules are very short and the overalllevel of microtubule content appears to be reduced. At the highestconcentration, cells often round up. FIG. 36 shows images ofmicrotubules in cells exposed to ACEA100108 for 24 hours. At both 1 uMand 3 uM, the cells seem to have made somewhat of a recovery between 4and 24 hours. The microtubule networks in both cases appear relativelynormal. However, at 9 uM, the microtubules appear short and the networkis abnormal.

FIG. 37 shows images of microtubules in cells exposed to ACEA100116 for4 hours. Remnants of the microtubule network remain at 1 uM, but at thetwo higher concentrations, microtubules appear very short and abnormal.Cells are not elongated but rather appear to round up in adose-dependent manner. FIG. 38 shows images of microtubules in cellsexposed to ACEA100116 for 24 hours. Cells treated with only 1 uMACEA100116 appear relatively normal; essentially all cells treated with3 uM or 9 uM were dead after 24 hours of exposure to ACEA100116.

FIGS. 39-41 show the effect of DBTS and organosulfur compounds ACEA100108 and ACEA100116 on microtubule assembly in vitro. As shown in FIG.39 a, all doses of DBTS inhibit the extent of microtubule assemblysignificantly. The effect is especially prominent at 9 uM. Microtubulestructure of both the control (FIG. 39 b) and drug treated (FIG. 39 c)samples were visualized by electron microscopy. As shown in FIG. 40,lower dosages of ACEA100108 had minimal effects upon microtubuleassembly. In contrast, 27 uM ACEA100108 had a marked inhibitory effectupon the extent of microtubule assembly.

As shown in FIG. 41, ACEA100116 is very different that DBTS andACEA100108. Whereas the other two drugs inhibit microtubule assembly,ACEA100116 promotes microtubule assembly. This is apparent at both 9 uMand 27 uM. This plot also exhibits a common, but not well understood,phenomena known as “overshooting” in which the light scattering patterndoes not plateau but rather steadily declines. Nonetheless, it is clearthat ACEA100116 promotes rather than inhibits microtubule assembly invitro.

Furthermore, DBTS, ACEA100108 and ACEA100116 were shown to influencemicrotubule behavior in vitro. As seen in Table 37, all three drugsaltered the pattern of microtubule dynamics. DBTS did not affect themicrotubule growth rate but did increase the average duration of growthevents and consequently the average length grown in a growth event. Italso increased the percentage of time spent growing. The average lengthof shortening events was also reduced.

ACEA100108 also increased the duration of growth events and the averagelength of growth events; it also had a strong effect upon the length ofshortening events; this effect was even more pronounced than that ofDBTS and ACEA100116 exhibited significantly different effects thaneither of the other two drugs. ACEA100116 increased the growth rate buthad little effect upon the length of growing events. It had no effectupon the rate of shortening, but had a strong effect upon the length ofshortening events. While the cell imaging data can not distinguishbetween the drugs binding to tubulin or microtubule associated proteins,the in vitro microtubule assembly and in vitro microtubule dynamicsassays both used only purified, MAP-free tubulin. These observationsdemonstrate that all three drugs interact directly with tubulin.

TABLE 37 DBTS and its derivative compounds ACEA100108 and ACEA100116suppress microtubule dynamics. Tubulin alone control DBTS .1uMACEA100108 .2uM ACEA 100116 .07uM Growth Rate ± SEM (um/min) 1.44 ± 0.11.55 ± 0.1  1.59 ± 0.1  1.79* ± 0.1  Length of Excursion (um) 2.34 ± 0.23.34 ± 0.3* 3.24 ± 0.3* 2.15 ± 0.2  Duration of Event (min) 1.63 ± 0.22.21 ± 0.3  2.04 ± 0.3  1.2 ± 0.2 % time spent in growth phase 31 45 3025 Shortening Rate ± SEM (um/min) 44.90 ± 12.6 35.40 ± 6.0  35.8 ± 4.3 42.0 ± 8.3  Length of Excursion (um) 10.54 ± 0.5  6.02 ± 0.3* 3.79 ±0.3* 4.55 ± 0.2* Duration of Event (min)  0.23 ± 0.06 0.17 ± 0.04 0.11 ±0.02 0.11 ± 0.02 % time spent in shortening phase  3  3  1  2 % timespent in attenuation phase 66 52 69 73 Mean duration of attenuation ±SEM 2.63 ± 0.3 2.15 ± 0.3  3.74 ± 0.7  2.22 ± 0.2  Frequency oftransitions ± SD (events/min) Catastrophes  0.12 ± 0.03 0.17 ± 0.04 0.09± 0.03 0.18 ± 0.04 Rescues  4.3 ± 1.1 5.3 ± 1.3 9.4 ± 3.0 8.7 ± 2.1Total  0.46 ± 0.06 0.50 ± 0.07 0.32 ± 0.06 0.61 ± 0.08 Dynamicity(um/min)    1.73    1.72    0.83    1.25 As of Mar. 31, 2005 *= p < 0.05or less

Example 24 ACEA100108 Induces Apoptosis in Cancer Cells

To test if ACEA100108 compound induces apoptosis in cancer cells, theA549 human lung cancer cells were treated with 1 uM ACEA100108 and 50 nMpaclitaxel or 10 nM vinblastine. Paclitaxel and vinblastine, the twosuppressors of microtubule dynamics were used as the positive control.A549 cells were seeded in chamber slides at a density of 10,000cells/well and 18 hours later were treated with the indicatedconcentrations of the anti-mitotic compounds ACEA100108, paclitaxel andvinblastine. The cells were incubated with the drugs for 24 hours andthen washed 2× with PBS and 3× with binding buffer (10 mM HEPES, pH 7.5,140 mM NaCl, 2.5 mM CaCl₂). The Cells were stained with 1 ug/mL AnnexinV-Cy3 conjugate (Red, staining the cells that are starting apoptoticprocess) and 500 uM 6-CFDA (Green, staining the viable cells) in 1×binding buffer for 20 minutes. The cells were gently washed 3× in 1×binding buffer, mounted, viewed under immunofluorescent microscope andimaged using an attached CCD camera. Note that live cells show stainingonly with 6-CFDA (green), while necrotic cells will stain only withAnnexin V-Cy3 (red). Cells starting the apoptotic process will stainboth with AnnexinV-Cy3 and 6-CFDA.

As shown in FIG. 42, the cells treated with ACEA100108, paclitaxel, andvinblastine showed strong staining of Annexin V, while the control cellswhich were only treated with DMSO showed no Annexin V staining. Thisindicates that ACEA100108 induces apoptosis in A549 human lung cancercells.

Example 25 ACEA100108 Induces G2/M Cell-Cycle Arrest in Cancer Cells

Microtubules are extremely important in the process of mitosis, duringwhich the duplicated chromosomes of a cell are separated into twoidentical sets before cleavage of the cell into two daughter cells.Compounds which target microtubules such as paclitaxel, and vinblastinesuppress the microtubule dynamics and block the process of mitosis. Asconsequence, cells will be arrested at G2/M phase. To test if ACEA100108influences the process of mitosis in cancer cell dividing, A549 humanlung cancer cells were treated with 25 uM ACEA100108 and 7.8 nMpaclitaxel, and the cell-cycle effects of the compounds were detected byflow cytometry.

In briefly, A549 cells were seeded at a density of 500,000 cells in 60mm tissue culture dishes. Approximately 18 hours later the cells weretreated with the indicated concentrations of anti-mitotic compounds andallowed to further incubate for 24 hours. The cells were washed in PBS,trypsinized, counted and fixed in ice-cold 70% methanol and stored at 4°C. The cells were washed with PBS, stained with propidium iodide andkept on ice until flow cytometry analysis. As shown in FIG. 43, the cellpopulation at G2/M phase increased significantly in cells treated withboth ACEA100108 and paclitaxel, compared with the cells treated withDMSO only.

Example 26 Large scale synthesis of Di(p-chlorobenzyl)trisulfide 9

N-Trimethylsilylimidazole (10.67 mL, 97%, d=0.956, actual weight=9.89 g,70.54 mmol) was dissolved in 70 mL of anhydrous hexanes in a dry 250-mLround-bottom flask. To this stirred solution was added slowly (40-50min) sulfur dichloride solution in dichloromethane (35.3 mL, 1.0 M, 35.3mmol) at room temperature under a nitrogen atmosphere. The whiteprecipitate was formed. The reaction mixture was stirred for 50 min, andthen cooled to 0° C. under a nitrogen atmosphere. A solution of4-chlorobenzyl mercaptan (9.5 mL, 96%, actual weight=11.19 g, 70.53mmol) in 50 mL of anhydrous hexanes was added dropwise under stirringand nitrogen atmosphere for 40-50 min. The resulting reaction mixturewas stirred at 0° C. for 1 hour, and then at room temperature for 3hours. The white to pale yellow solid was filtered off through a pad ofCelite and washed with small amount of hexanes. The filtrate was washedwith water (200 mL, 100 mL) and then saturated aqueous sodium chloridesolution (200 mL). The organic phase was dried over anhydrous sodiumsulfate. The drying agent was filtered off, and the filtrate wasconcentrated under reduced pressure.

The white solid residue was purified by flash chromatography on a silicagel column using hexanes-ethyl acetate (60:1) as an eluent. Thefractions were monitored with silica gel TLC using hexanes-ethyl acetate(40:1) as a developing solvent (R_(f)=0.45). The desired fractions werecollected, and the solvent was evaporated. The resulting white solidproduct was re-crystallized from hexanes to give 11.06 g (90%) desiredproduct 9 as white needle crystalline. ¹H NMR (499.1 MHz, CDCl₃) δ 3.98(s, 4H), 7.23 (d, 4H, J=8.4 Hz), 7.30 (d, 4H, J=8.4 Hz); ES MS m/z 345(M-1)⁻.

Example 27 Large scale synthesis of Di(p-fluorobenzyl)trisulfide 8

N-Trimethylsilylimidazole (21.42 mL, 97%, d=0.956, actual weight=19.86g, 141.6 mmol) was dissolved in 140 mL of anhydrous hexanes in a dry500-mL round-bottom flask. To this stirred solution was added slowly(40-50 min) sulfur dichloride solution in dichloromethane (70.8 mL, 1.0M, 70.8 mmol) at room temperature under a nitrogen atmosphere. The whiteprecipitate was formed. The reaction mixture was stirred for 50 min, andthen cooled to 0° C. under a nitrogen atmosphere. A solution of4-fluorobenzyl mercaptan (18.04 mL, 20.86 g, 96%, actual weight=20.0 g,140.8 mmol) in 100 mL of anhydrous hexanes was added dropwise understirring and nitrogen atmosphere for 40-50 min. The resulting reactionmixture was stirred at 0° C. for 1 hour, and then at room temperaturefor 3 hours. The white to pale yellow solid was filtered off through apad of Celite and washed with small amount of hexanes. The filtrate waswashed with water (400 mL, 300 mL) and then saturated aqueous sodiumchloride solution (400 mL). The organic phase was dried over anhydroussodium sulfate. The drying agent was filtered off, and the filtrate wasconcentrated under reduced pressure.

The white solid residue was purified by flash chromatography on a silicagel column using hexanes-ethyl acetate (60:1) as an eluent. Thefractions were monitored with silica gel TLC using hexanes-ethyl acetate(40:1) as a developing solvent (R_(f)=0.46). The desired fractions werecollected, and the solvent was evaporated. The resulting white solidproduct was re-crystallized from hexanes to give 14.7 g (67%) desiredproduct as white needle crystalline. The mother liquor was concentrated.Further re-crystallization provided 10-15% more crystalline product. m.p. 61.5-62.1° C.; UV-VIS λ=218 nm (ω, 63700), λ=283 nm (ω, 12000); ¹HNMR (499.1 MHz, CDCl₃) δ 4.00 (s, 4H), 7.01 (t, 4H, J=8.8 Hz), 7.27 (dd,4H, J=8.8, 5.4 Hz); ¹³C NMR (125.7 MHz, CDCl₃) δ 42.4, 115.6, 115.8,131.2, 131.3, 132.4, 162.5 (C—F, J=250 Hz); ¹⁹F NMR (376.5 MHz, CDCl₃) δ−114.2; ES MS m/z 337/338 (M+Na)⁺; Anal. Calcd. for C₁₄H₁₂F₂S₃: C,53.48; H, 3.85; S, 30.59. Found: C, 53.16; H, 4.22; S, 30.24.

Example 28 Large scale synthesis of di(p-fluorobenzyl)trisulfide (8)Using Pure Sulfur Dichloride

N-Trimethylsilylimidazole (226.6 mL, 97%, d=0.956, actual weight=205.7g, 1467 mmol) was dissolved in 1200 mL of anhydrous hexane and 560 mL ofanhydrous dichloromethane (dried with molecular sieves type 3A) in a dry3000-mL three-necked flask. To this stirred solution was added slowly(40-50 min) pure sulfur dichloride (55.9 mL, 90.63 g, 880 mmol, 0.6 eq)at room temperature under a nitrogen atmosphere. The reaction took placeimmediately with precipitate. The reaction mixture was stirred for 50min, and then cooled to 0° C. under a nitrogen atmosphere. A solution of4-fluorobenzyl mercaptan (176 mL, 96%, actual weight=200.17 g, 1408mmol) in 250 mL of anhydrous dichloromethane and 100 mL of anhydroushexane was added dropwise under stirring and nitrogen atmosphere for40-50 min. The resulting reaction mixture was stirred at 0° C. for 1hour, and then at room temperature for 3 hours. The reaction wasmonitored with TLC using hexane-ethyl acetate (40:1) as a developmentsolvent, and the result indicated that the reaction was normal andcompleted. The white to pale yellow solid was filtered off through a padof Celite and washed with small amount of hexane. The filtrate waswashed twice with water (1000 mL×2) and then once with saturated aqueoussodium chloride solution (1000 mL). The organic phase was dried overanhydrous sodium sulfate. The drying agent was filtered off, and thefiltrate was concentrated under reduced pressure. The crude product waspurified by flash chromatography on a silica gel column (8×36 cm) usingpetroleum ether (60-90° C. fraction)-ethyl acetate (80:1, 60:1, 40:1 andthen 20:1) as gradient eluents. The fractions were monitored with silicagel TLC using n-hexane-ethyl acetate (40:1) as a developing solvent(R_(f)=0.46). The desired fractions were collected, and the solvent wasevaporated. The resulting white solid product was re-crystallized from1000 mL of hexane to give 131.0 g of the desired product 8 as whiteneedle crystalline in 59.2% yield (T yield 221.16 g). m. p. 61.5-62.1°C.; UV-VIS λ=218 nm (ω, 63700), λ=283 nm (ω, 12000); ¹H NMR (499.1 MHz,CDCl₃) δ 4.00 (s, 4H), 7.01 (t, 4H, J=8.8 Hz), 7.27 (dd, 4H, J=8.8, 5.4Hz); ¹³C NMR (125.7 MHz, CDCl₃) δ42.4, 115.6, 115.8, 131.2, 131.3,132.4, 162.5 (C—F, J=250 Hz); ¹⁹F NMR (376.5 MHz, CDCl₃) δ −114.2; ES MSm/z 337/338 (M+Na)⁺; Anal. Calcd. for C₁₄H₁₂F₂S₃: C, 53.48; H, 3.85; S,30.59. Found: C, 53.16; H, 4.22; S, 30.24.

The asymmetric trisulfides 41-68 (Scheme 3) can be synthesized by MethodB similar to the reported procedure (Derbesy, G.; Harpp, D. N.Tetrahedron Letters, 1994, 35, 5381-5384). For example, a solution ofphenylthiol (C₆H₅CH₂SH) (10 mmol) and anhydrous pyridine (10 mmol) in 25mL of diethyl ether is added dropwise over a period of 30 minutes to acold (−78° C.) stirred solution of sulfur dichloride (10 mmol) in 50 mLof anhydrous diethyl ether. The reaction mixture is stirred for 30minutes. The corresponding second thiol (10 mmol) and anhydrous pyridine(10 mmol) in 25 mL of diethyl ether is added dropwise over a period of30 minutes at −78° C., and the reaction mixture is further stirred foran additional 30 minutes. The reaction mixture is washed with water (2times), 1 N sodium hydroxide solution (2 times), and then water (2times) until pH is neutral. The organic phase is dried over CaCl₂, oranhydrous sodium sulfate, filtered and concentrated. The residue ispassed through a short pad of silica gel using hexanes-ethyl acetate aseluent to provide high purity products 41-68 in 40-100% yields.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative, and are not to be takenas limitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications, including withoutlimitation those relating to the chemical structures, substituents,derivatives, intermediates, syntheses, formulations and/or methods ofuse of the invention, may be made without departing from the spirit andscope thereof. U.S. patents and publications referenced herein areincorporated by reference.

1. A compound having the formula:

wherein A and B are the same or different, A is benzene, pyridine,pyridazine, pyrimidine, pyrazine, triazine, isoxazole, isothiazole,oxadiazole, triazole, thiadiazole, pyrazole, imidazole, thiazole,oxazole, pyrrole, furan, thiophene indolizine, indole, isoindole,indoline, benzofuran, benzothiophene, indazole, benzimidazole,benzthiazole, purine, quinoxaline, quinoline, isoquinoline, cinnoline,phthalazine, quinazoline, quinoxaline, naphthyridine, pteridine,acridine, phenazine, phenothiazine, indene, or naphthalene, each ofwhich is optionally substituted with one or more heteroatoms selectedfrom O, N and S; halo; or C₁₋₁₀ alkyl, C₃₋₁₀ cyclic alkyl, C₂₋₁₀ alkenylor alkynyl, aryl, or heterocycle, each optionally containing one or moreheteroatoms; B is selected from pyridine, pyridazine, pyrimidine,pyrazine, triazine, isoxazole, isothiazole, oxadiazole, triazole,thiadiazole, pyrazole, imidazole, thiazole, oxazole, pyrrole, furan,thiophene indolizine, indole, isoindole, indoline, benzofuran,benzothiophene, indazole, benzimidazole, benzthiazole, purine,quinoxaline, quinoline, isoquinoline, cinnoline, phthalazine,quinazoline, quinoxaline, naphthyridine, pteridine, acridine, phenazine,phenothiazine, and indene, each of which is optionally substituted withone or more heteroatoms selected from O, N and S; halo; or C₁₋₁₀ alkyl,C₃₋₁₀ cyclic alkyl, C₂₋₁₀ alkenyl or alkynyl, aryl, or heterocycle, eachoptionally containing one or more heteroatoms; each S is optionally inthe form of an oxide; each R is H, halogen, carboxyl, cyano, amino,amido, an inorganic substituent, SR¹, OR¹ or R¹, wherein each R¹ isalkyl, alkenyl, alkynyl, aryl, heteroaryl, a carbocyclic ring or aheterocyclic ring, each of which is optionally substituted and maycontain a heteroatom; m, n and p are independently 0-3; or a compoundhaving formula (3) or (4):

wherein A, B, R, S, n and p are as defined above; or a pharmaceuticallyacceptable salt thereof.
 2. The compound of claim 1, wherein A and B arethe same.
 3. The compound of claim 1, wherein R is independently H,halo, OR¹, SR¹, CO₂R¹, CONR¹ ₂, C═O, CN, CF₃, OCF₃, NO₂, NR₁R₁, OCOR₁;or R is C₁₋₁₀ alkyl, C₃₋₁₀ cyclic alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,an aryl, heteroaryl, a carbocyclic ring or a heterocyclic ring, each ofwhich may contain a heteroatom.
 4. The compound of claim 1, wherein Aand B are independently selected from pyridine, pyridazine, pyrimidine,pyrazine, triazine, isoxazole, isothiazole, oxadiazole,[1,2,4]oxadiazole, triazole, thiadiazole, pyrazole, imidazole, thiazole,oxazole, benzoxazole, pyrrole, furan, thiophene indolizine, indole,isoindole, indoline, benzofuran, benzothiophene, indazole,benzimidazole, benzthiazole, purine, quinoxaline, quinoline,isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline,naphthyridine, pteridine, acridine, phenazine, phenothiazine, indene,benzoxadiazol, and benzo[1,2,5]-oxadiazole.
 5. The compound of claim 1,wherein A and B are independently selected from pyridine, pyridazine,pyrimidine, pyrazine, isoxazole, isothiazole, oxadiazole, thiazole,oxazole, benzoxazole, furan, thiophene indolizine, benzofuran,benzothiophene, and benzo[1,2,5]-oxadiazole.
 6. The compound of claim 1,wherein said compound has the formula (7)

wherein Ar is an optionally substituted thiophene, benzothiophene,pyridine or pyrazine.
 7. The compound of claim 1, which is a compound offormula (1) wherein p is
 1. 8. The compound of claim 1, wherein each nis 0 or
 1. 9. The compound of claim 1, wherein each R is H.
 10. A methodfor ameliorating or treating neuroblastoma, comprising administering toa subject in need thereof an effective amount of a compound in claim 1or a pharmaceutical composition thereof and optionally with anantiproliferative agent, whereby said neuroblastoma is ameliorated ortreated.
 11. A method for treating or ameliorating a cancer selectedfrom leukemia, lymphoma, lung cancer, colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer, breast cancer,head-neck cancer, pancreatic cancer, and renal cancer, comprisingadministering to a subject in need thereof an effective amount of thecompound of claim 1, or a pharmaceutical composition thereof, andoptionally with an antiproliferative agent; whereby said cellproliferative disorder in said subject is ameliorated or treated. 12.The method of claim 11, wherein said subject is human.
 13. The method ofclaim 11, wherein said compound is a compound according to claim
 7. 14.The method of claim 13, wherein each n is 0 or
 1. 15. The method ofclaim 11, wherein said compound is a compound according to claim
 6. 16.A method for ameliorating or treating restenosis, comprisingadministering to a subject in need thereof an effective amount of acompound of claim 1, or a pharmaceutical composition thereof, wherebyrestenosis in said subject is ameliorated or treated.
 17. The method ofclaim 16, wherein said restenosis is associated with neointimalhyperplasia.
 18. The method of claim 16, wherein said administering stepis oral or parental administration, or administration via a stent.
 19. Apharmaceutical composition comprising a compound of claim 1, and apharmaceutically acceptable excipient.
 20. A method for preparing acompound of claim 1, comprising: a) contacting N-trimethylsilylimidazole with sulfur dichloride in a halogenated solvent to providediimidazolylsulfide; and b) contacting said diimidazolylsulfide withmercaptan.
 21. A method for preparing a composition comprising acompound of claim 1, comprising: a) dissolving a compound of claim 1 ina water-soluble organic solvent, a non-ionic solvent, a water-solublelipid, a cyclodextrin, a vitamin, a fatty acid, a fatty acid ester, aphospholipid, or a combination thereof, to provide a solution; and b)adding saline or a buffer containing 1-0% carbohydrate solution.